Multicomponent vaccines

ABSTRACT

Multicomponent vaccines are provided which aid in the prevention and treatment of staphylococcal infections and which include certain selected combinations of bacterial binding proteins or fragments thereof, or antibodies to those proteins or fragments. By careful selection of the proteins, fragments, or antibodies, a vaccine is provided that imparts protection against a broad spectrum of Staphylococcus bacterial strains and against proteins that are expressed at different stages of the logarithmic growth curve. In one embodiment of the invention, a composition is provided that includes at least a collagen binding protein or peptide (or an appropriate site directed mutated sequence thereof) such as CNA, or a protein or fragment with sufficiently high homology thereto, in combination with a fibrinogen binding protein, preferably Clumping factor A (“ClfA”) or Clumping factor B (“ClfB”), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto. The vaccines and products of the present invention are advantageous in that they respond to the urgent need of the medical community for a substitute for small molecule antibiotics, which are rapidly losing effectiveness and provide effective combinations of the large number of known bacterial surface adhesins which can impart effective protection against a broad spectrum of bacterial infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisionalapplication Ser. No. 60/098,439, filed Aug. 31, 1998.

The present invention was made in part from work supported by grant no.97-35204-5046 from the United States Department of Agriculture. The U.S.government has certain rights in this invention.

The invention is in the field of biological products for the treatmentand diagnosis of bacterial infections.

BACKGROUND OF THE INVENTION

Staphylococci are Gram-positive spherical cells, usually arranged ingrape-like irregular clusters. Some are members of the normal flora ofthe skin and mucous membranes of humans, others cause suppuration,abscess formation, a variety of pyogenic infections, and even fatalsepticemia. Pathogenic staphylococci often hemolyze blood, coagulateplasma, and produce a variety of extracellular enzymes and toxins. Themost common type of food poisoning is caused by a heat-stablestaphylococci enterotoxin.

The genus Staphylococcus has at least 30 species. The three main speciesof clinical importance are Staphylococcus aureus, Staphylococcusepidermidis, and Staphylococcus saprophyticus. Staphylococcus aureus iscoagulase-positive, which differentiates it from the other species. S.aureus is a major pathogen for humans. Almost every person has some typeof S. aureus infection during a lifetime, ranging in severity from foodpoisoning or minor skin infections to severe life-threateninginfections. The coagulase-negative staphylococci are normal human florawhich sometimes cause infection, often associated with implanteddevices, especially in very young, old and immunocompromised patients.Approximately 75% of the infections caused by coagulase-negativestaphylococci are due to S. epidermidis. Infections due toStaphylococcus warneri, Staphylococcus hominis, and other species areless common. S. saprophyticus is a relatively common cause of urinarytract infections in young women. The staphylococci produce catalase,which differentiates them from the streptococci.

S. aureus colonization of the articular cartilage, of which collagen isa major component, within the joint space appears to be an importantfactor contributing to the development of septic arthritis.Hematogenously acquired bacterial arthritis remains a serious medicalproblem. This rapidly progressive and highly destructive joint diseaseis difficult to eradicate. Typically, less than 50% of the infectedpatients fail to recover without serious joint damage. S. aureus is thepredominant pathogen isolated from adult patients with hematogenous andsecondary osteomyelitis.

In hospitalized patients, Staphylococcus bacteria such as S. aureus area major cause of infection. Initial localized infections of wounds orindwelling medical devices can lead to more serious invasive infectionssuch as septicemia, osteomyelitis, mastitis and endocarditis. Ininfections associated with medical devices, plastic and metal surfacesbecome coated with host plasma and matrix proteins such as fibrinogenand fibronectin shortly after implantation. The ability of S. aureus andother staphylococcal bacteria to adhere to these proteins is essentialto the initiation of infection. Vascular grafts, intravenous catheters,artificial heart valves, and cardiac assist devices are thrombogenic andprone to bacterial colonization. Of the staphylococcal bacteria, S.aureus is generally the most damaging pathogen of such infections.

A significant increase in S. aureus isolates that exhibit resistance tomost of the antibiotics currently available to treat infections has beenobserved in hospitals throughout the world. The development ofpenicillin to combat S. aureus was a major advance in infection controland treatment. Unfortunately, penicillin-resistant organisms quicklyemerged and the need for new antibiotics was paramount. With theintroduction of every new antibiotic, S. aureus has been able to counterwith β-lactamases, altered penicillin-binding proteins, and mutated cellmembrane proteins allowing the bacterium to persist. Consequently,methicillin-resistant S. aureus (MRSA) and multidrug resistant organismshave emerged and established major footholds in hospitals and nursinghomes around the world. (Chambers, H. F., Clin Microbiol Rev, 1:173,1988; and Mulligan, M. E., et al., Am J Med, 94:313, 1993) Today, almosthalf of the staphylococcal strains causing nosocomial infections areresistant to all antibiotics except vancomycin, and it appears to beonly a matter of time before vancomycin will become ineffective as well.

There is a strong and rapidly growing need for therapeutics to treatinfections from staphylococci such as S. aureus which are effectiveagainst antibiotic resistant strains of the bacteria. The U.S. NationalInstitutes for Health has recently indicated that this goal is now anational priority.

MSCRAMMs

Bacterial adherence to host tissue occurs when specific microbialsurface adhesins termed MSCRAMMs (Microbial Surface ComponentsRecognizing Adhesive Matrix Molecules) specifically recognize and bindto extracellular matrix (ECM) components, such as fibronectin,fibrinogen, collagen, and elastin. Many pathogenic bacteria have beenshown to specifically recognize and bind to various components of theECM in an interaction which appears to represent a host tissuecolonization mechanism. This adherence involves a group of bacterialproteins termed MSCRAMMs (Patti, J., et al., Ann Rev Microbiol,48:585-617, 1994; Patti, J. and Hook, M., Cur Opin Cell Biol.,6:752-758, 1994).

MSCRAMMs on the bacterial cell surface and ligands within the hosttissue interact in a lock and key fashion resulting in the adherence ofbacteria to the host. Adhesion is often required for bacterial survivaland helps bacteria evade host defense mechanisms and antibioticchallenges. Once the bacteria have successfully adhered and colonizedhost tissues, their physiology is dramatically altered and damagingcomponents such as toxins and enzymes are secreted. Moreover, theadherent bacteria often produce a biofilm and quickly become resistantto the killing effect of most antibiotics.

A bacterium can express MSCRAMMs that recognize a variety of matrixproteins. Ligand-binding sites in MSCRAMMs appear to be defined byrelatively short contiguous stretches of amino acid sequences (motifs).Because a similar motif can be found in several different species ofbacteria, it appears as though these functional motifs are subjected tointerspecies transfer (Patti and Hook, Curr Opin Cell Biol, 6:752-758,1994). In addition, a single MSCRAMM can sometimes bind several ECMligands.

Vaccination Studies

Historically, studies on bacterial adherence have focused primarily onGram-negative bacteria, which express a wide variety of fimbrialadhesive proteins (designated adhesins) on their cell surface (Falkow,S., Cell, 65:1099-1102, 1991). These adhesins recognize specificglycoconjugates exposed on the surface of host cells (particularlyepithelial layers). Employing the lectin-like structures in attachmentallows the microorganism to efficiently colonize the epithelialsurfaces. This provides the bacteria an excellent location forreplication and also the opportunity to disseminate to neighboring hosttissues. It has been demonstrated that immunization with pilus adhesinscan elicit protection against microbial challenge, such as in Hemophilusinfluenza induced otitis media in a chinchilla model (Sirakova et al.,Infect Immun, 62(5):2002-2020, 1994), Moraxella bovis in experimentallyinduced infectious bovine keratoconjunctivitis (Lepper et al., VetMicrobiol, 45(2-3):129-138, 1995), and E. coli induced diarrhea inrabbits (McQueen et al., Vaccine, 11:201-206, 1993). In most cases,immunization with adhesins leads to the production of immune antibodiesthat prevent infection by inhibiting bacterial attachment andcolonization, as well as enhancing bacterial opsonophagocytosis andantibody-dependent complement-mediated killing.

The use of molecules that mediate the adhesion of pathogenic microbes tohost tissue components as vaccine components is emerging as an importantstep in the development of future vaccines. Because bacterial adherenceis the critical first step in the development of most infections, it isan attractive target for the development of novel vaccines. An increasedunderstanding of the interactions between MSCRAMMs and host tissuecomponents at the molecular level coupled with new techniques inrecombinant DNA technology have laid the foundation for a new generationof subunit vaccines. Entire or specific domains of MSCRAMMs, either intheir native or site-specifically altered forms, can now be produced.Moreover, the ability to mix and match MSCRAMMs from differentmicroorganisms creates the possibility of designing a single vaccinethat will protect against multiple bacteria.

Recent clinical trials with a new subunit vaccine against whoopingcough, consisting of the purified Bordatella pertussis MSCRAMMsfilamentous hemagglutinin and pertactin, in addition to an inactivatedpertussis toxin, are a prime example of the success of this type ofapproach. Several versions of the new acellular vaccine were shown to besafe and more efficacious than the old vaccine that contained wholebacterial cells (Greco et al., N Eng J Med, 334:341-348, 1996;Gustaffson et al., N Eng J Med, 334:349-355, 1996).

Natural immunity to S. aureus infections remains poorly understood.Typically, healthy humans and animals exhibit a high degree of innateresistance to S. aureus infections. Protection is attributed to intactepithelial and mucosal barriers and normal cellular and humoralresponses. Titers of antibodies to S. aureus components are elevatedafter severe infections (Ryding et al., J Med Microbiol, 43(5):328-334,1995), however to date there is no serological evidence of a correlationbetween antibody titers and human immunity.

Over the past several decades live, heat-killed, and formalin fixedpreparations of S. aureus cells have been tested as vaccines to preventstaphylococcal infections. A multicenter clinical trial was designed tostudy the effects of a commercial vaccine, consisting of astaphylococcus toxoid and whole killed staphylococci, on the incidenceof peritonitis, exit site infection, and S. aureus nasal carriage amongcontinuous peritoneal dialysis patients (Poole-Warren et al., ClinNephrol., 35:198-206, 1991). Although immunization with the vaccineelicited an increase in the level of specific antibodies to S. aureus,the incidence of peritonitis was unaffected. Similarly, immunization ofrabbits with whole cells of S. aureus could not prevent or modify anystage in the development of experimental endocarditis, reduce theincidence of renal abscess, or lower the bacterial load in infectedkidneys (Greenberg, D. P., et al., Infect Immun, 55:3030-3034, 1987).

Currently there is no FDA approved vaccine for the prevention of S.aureus infections. However, a S. aureus vaccine (StaphVAX), based oncapsular polysaccharide, is currently being developed by NABI (NorthAmerican Biologicals Inc.). This vaccine consists of type 5 or type 8capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A(rEPA). The vaccine is designed to induce type-specific opsonicantibodies and enhance opsonophagocytosis (Karakawa et al., InfectImmun, 56:1090-1095, 1988). Using a refined lethal challenge mouse model(Fattom et al., Infect Immun, 61:1023-1032, 1993) it has been shown thatintraperitoneal infusion of type 5 capsular polysaccharide specific IgGreduces the mortality of mice inoculated intraperitoneally with S.aureus. The type 5 capsular polysaccharide-rEPA vaccine has also beenused to vaccinate seventeen patients with end-stage renal disease (Welchet al., J Amer Soc Nephrol, 7(2):247-253, 1996). Geometric mean (GM) IgGantibody levels to the type 5 conjugate increased between 13 and 17-foldafter the first immunization, however no additional increases could bedetected after additional injections. Interestingly, the GM IgM levelsof the vaccinated patients were significantly lower than controlindividuals. Supported by the animal studies, the vaccine has recentlycompleted a Phase II trial in continuous ambulatory peritorneal dialysispatients. The clinical trial showed the vaccine to be safe butineffective in preventing staphylococcal infections (NABI SEC FORM10-K405, Dec. 31, 1995). Two possible explanations for the inability ofStaph VAX to prevent infections related to peritoneal dialysis invaccinated patients are that the immunogenicity of the vaccine was toolow due to suboptimal vaccine dosing or that antibodies in thebloodstream are unable to affect infection in certain anatomic areas,such as the peritoneum.

Gram-positive bacteria related sepsis is on the increase. In factbetween one-third and one-half of all cases of sepsis are caused byGram-positive bacteria, particularly S. aureus and S. epidermidis. Inthe United States, it can be estimated that over 200,000 patients willdevelop Gram-positive related sepsis this year.

Using a mouse model (Bremell et al., Infect Immun. 59(8):2615-2623,1991), it has been clearly demonstrated that active immunization withM55 domain of the Col-binding MSCRAMM can protect mice against sepsisinduced death. Mice were immunized subcutaneously with either M55 or acontrol antigen (bovine serum albumin) and then challenged intravenouslywith S. aureus. Eighty-three percent (35/42) of the mice immunized withM55 survived compared to only 27% of the BSA immunized mice (12/45).This a compilation of 3 separate studies.

Schennings, et al, demonstrated that immunization with fibronectinbinding protein from S. aureus protects against experimentalendocarditis in rats (Micro Pathog, 15:227-236, 1993). Rats wereimmunized with a fusion protein (gal-FnBP) encompassingbeta-galactosidase and the domains of fibronectin binding protein fromS. aureus responsible for binding to fibronectin. Antibodies againstfusion protein gal-FnBP were shown to block the binding of S. aureus toimmobilized fibronectin in vitro. Endocarditis in immunized andnon-immunized control rats was induced by catheterization via the rightcarotid artery, resulting in damaged aortic heart valves which becamecovered by fibrinogen and fibronectin. The catheterized rats were theninfected intravenously with 1×10 5 cells of S. aureus. The number ofbacteria associated with aortic valves was determined 11/2 days afterthe challenge infection and a significant difference in bacterialnumbers between immunized and non-immunized groups was then observed.

A mouse mastitis model was used by Mamo, et al., (Vaccine, 12:988-992,1994) to study the effect of vaccination with fibrinogen bindingproteins (especially FnBP-A) and collagen binding protein from S. aureusagainst challenge infection with S. aureus. The mice vaccinated withfibrinogen binding proteins showed reduced rates of mastitis comparedwith controls. Gross examination of challenged mammary glands of miceshowed that the glands of mice immunized with fibrinogen bindingproteins developed mild intramammary infection or had no pathologicalchanges compared with glands from control mice. A significantly reducednumber of bacteria could be recovered in the glands from mice immunizedwith fibrinogen binding proteins as compared with controls. Mamo thenfound that vaccination with FnBP-A combined with staphylococcal alphatoxoid did not improve the protection (Mamo, et al., Vaccine,12:988-992, 1994). Next, Mamo, et al., immunized mice with only collagenbinding protein, which did not induce protection against the challengeinfection with S. aureus.

Whole killed staphylococci were included in a vaccine study in humansundergoing peritoneal dialysis (Poole-Warren et al., Clin. Nephrol,35:198-206, 1991). In this clinical trial, a commercially availablevaccine of alpha-hemolysin toxoid combined with a suspension of wholekilled bacteria) was administered intramuscularly ten times over 12months, with control patients receiving saline injections. Vaccinationelicited significant increases in the levels of antibodies to S. aureuscells in the peritoneal fluid and to alpha-hemolysin in the serum.However, immunization did not reduce the incidences of peritonitis,catheter-related infections or nasal colonization among vaccinerecipients. The lack of protective efficacy in this trial wereattributed to a suboptimal vaccine formulation.

Secreted proteins have been explored as components of subcellularvaccines. The alpha toxin is among the most potent staphylococcalexotoxins; it has cytolytic activity, induces tissue necrosis and killslaboratory animals. Immunization with formaldehyde-detoxified alphatoxin does not protect animals from systemic or localized infections,although it may reduce the clinical severity of the infections (Ekstedt,R. D., in The Staphylococci, 385-418, 1972).

One study has evaluated the protective efficacy of antibodies to the S.aureus microcapsule in an experimental model of staphylococcal infection(Nemeth, J. and Lee, J. C., Infect. Immun. 63:375-380, 1995). Rats wereactively immunized with killed, microencapsulated bacteria or passivelyimmunized with high-titer rabbit antiserum specific for the capsularpolysaccharide. Control animals were injected with saline or passivelyimmunized with normal rabbit serum. Protection against catheter-inducedendocarditis resulting from intravenous challenge with the same strainwas then evaluated. Despite having elevated levels of anticapsularantibodies, the immunized animals were susceptible to staphylococcalendocarditis and immunized and control animals had similar numbers ofbacteria in the blood.

As described in the Detailed Description of the Invention hereinbelow, anumber of patents and patent applications describe the gene sequencesfor fibronectin, fibrinogen, collagen, elastin, and MHC II analogoustype binding proteins. These patents and patent applications areincorporated by reference in their entirety. These documents teach thatthe proteins, fragments, or antibodies immunoreactive with thoseproteins or fragments can be used in vaccinations for the treatment ofS. aureus infections. PCT/US97/087210 discloses the vaccination of micewith a combination of a collagen binding protein (M55 fragment), afibronectin binding peptide (formulin treated FnBP-A (D1-D3)) and afibrinogen binding peptide (ClfA).

The lack of adequate protection against staphylococcal infection thathas been seen to date from the vaccines described above is likely theresult of the failure to generate the proper immune response, perhapsalong with improper immunization scheduling or an improper immunizationroute. Additional factors that also contribute to the poor performanceof past vaccines can be reflected in the fact that staphylococcalbacteria such as S. aureus have been observed to temporally regulate theexpression of most of its virulence factors via regulatory genes lociagr and sar. For example, S. aureus contains two genes that encode cellsurface fibrinogen binding proteins, ClfA and ClfB. Interestingly, ClfAis predominately expressed in early exponential growth, while ClfB isexpressed later in the growth phase. Accordingly, the antigens that theinvading organism presents to the host in vivo may not be the same asthose used in the vaccine. In addition, not every S. aureus antigen isexpressed on every isolate. For example, only about 50% of S. aureusclinical isolates express the gene cna, which encodes for the collagenbinding MSCRAMM. To generate an effective immunotherapeutic against S.aureus, the vaccine must be multi-component and contain antigens thatspan the growth cycle as well as include antigens that are expressed bya majority of S. aureus isolates.

Despite the advances in the art of compositions for the treatment ofinfections from staphylococcal bacteria such as S. aureus, there remainsa need to provide a more effective product, and preferably one thatexhibits a broad spectrum immunization against staphylococcal bacteriaof various strains, and to particular proteins which may be expressed atdifferent stages of the bacterial growth phase.

Therefore, it is an object of the invention to provide a new therapeuticcomposition for immunization against infections from staphylococcalbacteria such as S. aureus and S. epidermidis.

It is another object of the present invention to provide a vaccine thatwill provide protection against mastitis, arthritis, endocarditis,septicemia, and osteomyelitis, furunculosis, cellulitis, pyemia,pneumonia, pyoderma, supporation of wounds, food poisoning, bladderinfections and other infectious diseases.

It is another object of the present invention to provide a therapeuticcomposition that immunizes against staphylococcal infection, enhancesthe amount of intracellular killing of staphylococcal bacteria, andincreases the rate of phagocytosis of staphylococcal bacteria.

It is still another object of the present invention to provide acomposition that will further protect the host by neutralizingexotoxins.

SUMMARY OF THE INVENTION

It has been discovered that the treatment of staphylococcal infectionscan be significantly enhanced by immunization with certain selectedcombinations of bacterial binding proteins or fragments thereof, orantibodies to those proteins or fragments. The proteins or fragments canbe used in active vaccines, and the antibodies in passive vaccines.Alternatively, the combinations can be used to select donor blood poolsfor the preparation of purified blood products for passive immunization.By careful selection of the proteins, fragments, or antibodies, avaccine is provided that imparts protection against a broad spectrum ofStaphylococcus bacterial strains and against proteins that are expressedat different stages of the logarithmic growth curve.

The vaccine and products described herein respond to the urgent need ofthe medical community for a substitute for small molecule antibiotics,which are rapidly losing effectiveness. The vaccines are a significantimprovement over the prior art, which while generally teaching the useof MSCRAMMs to impart immunization, did not teach which combinations ofthe large number of known MSCRAMMs should be used to impart superiorprotection.

In one embodiment of the invention, a composition is provided thatincludes at least a collagen binding protein or peptide (or anappropriate site directed mutated sequence thereof) such as CNA, or aprotein or fragment with sufficiently high homology thereto, incombination with a fibrinogen binding protein, preferably Clumpingfactor A (“ClfA”) or Clumping factor B (“ClfB”), or a useful fragmentthereof or a protein or fragment with sufficiently high homologythereto.

In another embodiment of the invention, a composition is provided thatincludes at least a fibronectin binding protein or peptide (or anappropriate site directed mutated sequence thereof), or a protein orfragment with sufficiently high homology thereto, in combination withthe fibrinogen binding protein, preferably A or B (ClfA or ClfB,respectively), or a useful fragment thereof or a protein or fragmentwith sufficiently high homology thereto.

In a third embodiment, a composition is provided that includes at leastthe fibrinogen binding protein A (ClfA) and the fibrinogen bindingprotein B (ClfB), or useful fragments thereof or a protein or fragmentwith sufficiently high homology thereto.

In a fourth embodiment, a composition is provided that includes at leasta fibronectin binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), or a protein or fragment withsufficiently high homology thereto, in combination with (i) thefibrinogen binding protein A and B (ClfA and ClfB), or a useful fragmentthereof or a protein or fragment with sufficiently high homologythereto; and (ii) a collagen binding protein or useful fragment thereof.

In an additional embodiment, a composition is provided that includes thecomponents of the prior embodiments in combination with an elastinbinding protein or peptide or a protein or fragment with sufficientlyhigh homology thereto.

In another embodiment, a composition is provided that includes thecomponents of the prior embodiments in combination with a MHC IIanalogous protein or peptide or a protein or fragment with sufficientlyhigh homology thereto.

In another embodiment, a composition is provided that includes thecomponents of any of the prior combinations in combination with abacterial component to increase the rate of phagocytosis of thestaphylococcal bacteria. In a one such embodiment, the bacterialcomponent comprises a capsular polysaccharide, such as capsularpolysaccharide type 5 or type 8.

In an additional embodiment, a composition is provided that includes anyof the prior combinations in combination with the extracellularmatrix-binding proteins SdrC, SdrD, SdrE or a consensus or variablesequence amino acid motif, or useful fragments thereof or proteins orfragments with sufficiently high homology thereto.

In an additional embodiment, a composition is provided that includes andof the prior combinations in combination with the extracellularmatrix-binding proteins SdrF, SdrG, or SdrH, or a consensus or variablesequence amino acid motif, or useful fragments thereof or proteins orfragments with sufficiently high homology thereto. This embodiment isparticularly effective in developing vaccines that can be useful withregard to both coagulase-positive and coagulase-negative staphylococcalbacteria.

In another embodiment, a composition is provided that includes at leastthe extracellular matrix-binding proteins SdrC, SdrD and SdrE or usefulfragments thereof, such as the consensus or variable sequence amino acidmotif, or a protein or fragment with sufficiently high homology thereto.

Alternatively, compositions are provided that include monoclonal orpolyclonal antibodies which are immunoreactive to the selectedcombination of described components. These compositions can be used invaccinations to treat patients infected with Staphylococcus infections.

In other embodiments of the invention, the combinations of proteins,fragments or antibodies as described are used in diagnostic kits.

As described below, proteins and peptides to be used in the compositionwhich bind to fibronectin, fibrinogen, collagen, and elastin are known.Alternatively, one can identify new fibronectin, fibrinogen, collagen,and elastin binding proteins, or the epitopes thereof for use in thecomposition. Methods of identifying a peptide of a binding domain of abinding protein that binds to the ligand of choice are known. Forexample, one can contact a candidate protein or peptide with the ligandunder conditions effective to allow binding of the ligand to the bindingdomain of a binding protein, and identify a positive candidate peptidethat binds to the ligand.

Antibodies that bind to the binding domains of the composition proteinsor peptides can be generated by administering to an animal apharmaceutical composition comprising an immunologically effectiveamount of the combination of proteins or peptides, even though thepeptide does not specifically bind to the ECM.

The combination of the isolated, recombinant or synthetic MSCRAMMproteins, or active fragments thereof or fusion proteins thereof, arealso useful as scientific research tools to identify staphylococcalbinding sites on the host ECM molecules, thereby promoting anunderstanding of the mechanisms of bacterial pathology and thedevelopment of antibacterial therapies. Furthermore, the isolated,recombinant or synthetic proteins, or antigenic portions thereof(including epitope-bearing fragments), or fusion proteins thereof can beadministered to animals as immunogens or antigens, alone or incombination with an adjuvant, for the production of antisera reactivewith MSCRAMM proteins. In addition, the proteins can be used to screenantisera for hyperimmune patients from whom can be derived antibodieshaving a very high affinity for the proteins. Antibodies isolated fromthe antisera are useful for the specific detection of staphylococcalbacteria or binding proteins, as research tools, or as therapeutictreatments against staphylococcal infection.

The proteins, or active fragments thereof, and antibodies to theproteins are useful for the treatment of infections from staphylococcalinfections from bacteria such as S. aureus as described above; for thedevelopment of anti-Staphylococcus vaccines for active or passiveimmunization; and, when administered as pharmaceutical composition to awound or used to coat medical devices or polymeric biomaterials in vitroand in vivo, both the proteins and the antibodies are useful as blockingagents to prevent or inhibit the binding of staphylococcal bacteria tothe wound site or biomaterials.

Preferably, animal derived antibody is modified so that it is lessimmunogenic in the patient to whom it is administered. For example, ifthe patient is a human, the antibody may be “humanized” by transplantingthe complimentarily determining regions of the hybridoma-derivedantibody into a human monoclonal antibody as described by Jones et al,(Nature 321:522-525 (1986)) or Tempest et al. (Biotechnology 9:266-273(1991)).

Kits are also provided that are useful as a diagnostic agent for thedetection of staphylococcal infections. According to yet anotherembodiment, anti-MSCRAMM antibodies as well as the MSCRAMM polypeptidesof this invention, are useful as diagnostic agents for detectinginfection by staphylococcal bacteria, because the polypeptides arecapable of binding to antibody molecules produced in animals, includinghumans that are infected with staphylococcal bacteria such as S. aureus,and the antibodies are capable of binding to particular staphylococcalbacteria or antigens thereof.

Diagnostic agents may be included in a kit which can also includeinstructions for use and other appropriate reagents. The kit can alsocontain a means to evaluate the product of the assay, for example, acolor chart, or numerical reference chart. The polypeptide or antibodymay be labeled with a detection means that allows for the detection ofthe MSCRAMM polypeptide when it is bound to an antibody, or for thedetection of the anti-MSCRAMM polypeptide antibody when it is bound toStaphylococcus bacteria.

The detection means may be a fluorescent labeling agent such asfluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and thelike, an enzyme, such as horseradish peroxidase (HRP), glucose oxidaseor the like, a radioactive element such as ¹²⁵I or ⁵¹Cr that producesgamma ray emissions, or a radioactive element that emits positrons whichproduce gamma rays upon encounters with electrons present in the testsolution, such as ¹¹C, ¹⁵O, or ¹³N. The linking of the detection meansis well known in the art. For instance, monoclonal anti-MSCRAMMpolypeptide antibody molecules produced by a hybridoma can bemetabolically labeled by incorporation of radioisotope-containing aminoacids in the culture medium, or polypeptides may be conjugated orcoupled to a detection means through activated functional groups.

The diagnostic kits of the present invention may be used to detect thepresence of a quantity of Staphylococcus bacteria or anti-Staphylococcusantibodies in a body fluid sample such as serum, plasma or urine. Thus,in preferred embodiments, an MSCRAMM polypeptide or anti-MSCRAMMpolypeptide antibody composition of the present invention is bound to asolid support typically by adsorption from an aqueous medium. Usefulsolid matrices are well known in the art, and include crosslinkeddextran; agarose; polystyrene; polyvinylchloride; cross-linkedpolyacrylamide; nitrocellulose or nylon-based materials; tubes, platesor the wells of microtiter plates. The polypeptides or antibodies of thepresent invention may be used as diagnostic agents in solution form oras a substantially dry powder, e.g., in lyophilized form.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a schematic representation of the peptides used inillustrative vaccine, MSCRAMM IV. This drawing illustrates the essentialfeatures of the collagen binding MSCRAMM CNA, fibrinogen binding MSCRAMMClfA, fibrinogen binding MSCRAMM ClfB and fibronectin binding MSCRAMMFnBPA proteins. The MSCRAMMs are shown with regions denoted that wereexpressed as recombinant proteins and used to generate antibodies inrabbits immunized with MSCRAMM IV. All proteins were designed with anamino terminal histidine tag to facilitate purification by metalchelating chromatography.

FIG. 2 is a time course graph of the immune response in MCSCRAMMvaccinated Rhesus Monkeys as shown by changes in antibody titers againstthe MSCRAMMs CNA, ClfA, ClfB and FnBPA, respectively. The titers wereanalyzed by ELISA and measured as changes in absorbance (quantified at405 nm) during each week over the course of a six-month period oftreatment following the original immunization with the antigen.

FIG. 3 shows the nucleic acid sequence coding for the sdrF gene from S.epidermidis, identified as SEQ ID NO: 1 and the amino acid sequencecoded thereby, identified as SEQ ID NOS: 2-6.

FIG. 4 shows the nucleic acid sequence coding for the sdrG gene from S.epidermidis, identified as SEQ ID NO: 7 and the amino acid sequencecoded thereby, identified as SEQ ID NOS: 8-12.

FIG. 5 shows the nucleic acid sequence coding for the sdrH gene from S.epidermidis, identified as SEQ ID NO: 13 and the amino acid sequencecoded thereby, identified as SEQ ID NO: 14.

DETAILED DESCRIPTION OF THE INVENTION

Compositions suitable for use as vaccines are provided that include atleast:

(i) A collagen binding protein, peptide or domain (or an appropriatesite directed mutated sequence thereof) such as CNA, or a protein,fragment or domain with sufficiently high homology thereto, incombination with a fibrinogen binding protein, preferably Clumpingfactor A (“ClfA”) or Clumping factor B (“ClfB”), or a useful fragmentthereof or a protein or fragment with sufficiently high homologythereto;

(ii) a fibronectin binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), or a protein or fragment withsufficiently high homology thereto, in combination with the fibrinogenbinding proteins A and B (ClfA and ClfB), or useful fragments thereof orproteins or fragments with sufficiently high homology thereto; or

(iii) the fibrinogen binding protein A (ClfA) and the fibrinogen bindingprotein B (ClfB), or useful fragments thereof or a protein or fragmentwith sufficiently high homology thereto; or

(iv) fibronectin binding protein or peptide (or an appropriate sitedirected mutated sequence thereof), or a protein or fragment withsufficiently high homology thereto, in combination with the fibrinogenbinding protein A and B (ClfA and ClfB), or a useful fragment thereof ora protein or fragment with sufficiently high homology thereto; and acollagen binding protein or useful fragment thereof, or a protein orfragment with sufficiently high homology thereto;

(v) components of any of the above embodiments in combination with anelastin binding protein or peptide or a protein or fragment withsufficiently high homology thereto; or

(vi) components of any of the above embodiments in combination with aMHC II analogous type binding protein or peptide, protein or fragmentwith sufficiently high homology thereto; or

(vi) components of any of the above embodiments in combination with abacterial component to increase the rate of phagocytosis of astaphylococcal bacteria such as S. aureus; or

(vii) components of any of the above embodiments in combination with theextracellular matrix-binding proteins SdrC, SdrD or SdrE, or usefulfragments thereof, such as a consensus or variable sequence amino acidmotif, or proteins or fragments with sufficiently high homology thereto;or

(viii) components of any of the above embodiments in combination withthe extracellular matrix-binding proteins SdrF, SdrG or SdrH, or usefulfragments thereof, such as a consensus or variable sequence amino acidmotif, or proteins or fragments with sufficiently high homology thereto,such that a vaccine created from said components will also be useful toimmunize a patient against infection from coagulase-negative bacteriasuch as S. epidermidis as well as coagulase positive bacteria such as S.aureus; or

(ix) the extracellular matrix-binding proteins SdrC, SdrD and SdrE oruseful fragments thereof, such as a consensus or variable sequence aminoacid motif, or a protein or fragment with sufficiently high homologythereto.

Isolated protein fragments from wild-type or naturally occurringvariants or synthetic or recombinant peptides corresponding towild-type, naturally occurring variants or introduced mutations that donot correspond to a naturally occurring binding domain of a bindingprotein can be used in these embodiments.

The isolated peptides should be of a sufficient length to allow for thegeneration of an antibody that binds both to the isolated peptide andthe binding domain, and blocks the binding of the binding protein to itsligand. In certain aspects, peptides comprising at least about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, about 20,about 22, about 24, about 25, about 30, about 35, about 40, about 45 orabout 50 contiguous amino acids are preferred. In other preferredaspects of the invention, the isolated peptide comprises at least about6 contiguous amino acids from the wild type sequence of the bindingdomain.

In one aspect of the invention, the isolated peptide or antibodycompositions are used to generate an immunological response in ananimal. In this aspect, the compositions preferably further comprise anadjuvant. Many adjuvants are known for use in vaccinations and arereadily adapted to this composition. The isolated peptide or proteincomposition is preferably dispersed in a pharmaceutically acceptableexcipient.

The isolated peptide can be linked to a selected amino acid sequence tomake a fusion protein. As a nonlimiting example, a fusion protein can bemade that comprises at least a first peptide of a binding domain of abinding protein operatively linked to a selected amino acid sequence. Inone embodiment, if the peptide is a fibronectin binding domain, thefirst peptide does not specifically bind to fibronectin. In preferredaspects, the first peptide is linked to a selected carrier molecule oramino acid sequence, including, but not limited to, keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA).

Immunological compositions, including vaccine, and other pharmaceuticalcompositions containing the selected MSCRAMM proteins or the DNAencoding such MSCRAMM proteins are included within the scope of thepresent invention. The combination of binding proteins, or active orantigenic fragments thereof, or fusion proteins thereof can beformulated and packaged, alone or in combination with other antigens,using methods and materials known to those skilled in the art forvaccines. The immunological response may be used therapeutically orprophylactically and may provide antibody immunity or cellular immunitysuch as that produced by T lymphocytes such as cytotoxic T lymphocytesor CD4+ T lymphocytes.

Vaccines can be prepared for use in both active and passiveimmunizations. Preferably the antigenic material is extensively dialyzedto remove undesired small molecular weight molecules and/or lyophilizedfor more ready formulation into a desired vehicle.

I. Definitions

The terms FnBP-A protein, FnBP-B protein, ClfA protein, ClfB protein,SdrC protein, SdrD protein, SdrE protein, SdrF protein, SdrG protein,SdrH protein, CNA protein, EbpS protein and MHCII protein are definedherein to include FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, SdrF,SdrG, SdrH, CNA, EbpS; and MHCII subdomains, respectively, active orantigenic fragments of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE,SdrF, SdrG, SdrH, CNA, EbpS and MHCII proteins, and proteins orfragments that have sufficiently high homology therewith. Activefragments of FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG,SdrH, CNA, EbpS and MHCII proteins are defined herein as peptides orpolypeptides capable of blocking the binding of Staphylococcus bacteriato host ECM. Antigenic fragments of FnBP-A, FnBP-B, ClfA, ClfB, SdrC,SdrD, SdrE, SdrF, SdrG, SdrH, CNA, EbpS and MHCII proteins are definedherein as peptides or polypeptides capable of producing an immunologicalresponse.

The term “adhesin” as used herein includes naturally occurring andsynthetic or recombinant proteins and peptides which can bind toextracellular matrix proteins and/or mediate adherence to host cells.

The term “amino acid” as used herein includes naturally occurring andsynthetic amino acids and includes, but is not limited to, alanine,valine, leucine, isoleucine, proline, phenylalanine, tryptophan,methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine,glutamate, aspartic acid, glutamic acid, lysine, arginine, andhistidine.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term as used herein includesmonoclonal antibodies, polyclonal, chimeric, single chain, bispecific,simianized, and humanized-antibodies as well as Fab fragments, includingthe products of an Fab immunoglobulin expression library.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

As used herein, an “antigenically functional equivalent” protein orpeptide is one that incorporates an epitope that is immunologicallycross-reactive with one or more epitopes either derived from any of theparticular MSCRAMM proteins disclosed (e.g., FnB-B, FnB-A, FnBP-B andFnBP-A) or derived from any of the particular bacterial componentsdisclosed (e.g., teichoic acids, alpha toxin and capsular polysaccharidetype 5). Antigenically functional equivalents, or epitopic sequences,may be first designed or predicted and then tested, or may simply bedirectly tested for cross-reactivity.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

A “cell line” is a clone of a primary cell that is capable of stablegrowth in vitro for many generations.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thesequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, prokaryotic sequences, cDNAfrom eukaryotic MRNA, genetic DNA sequences from eukaryotic (e.g.,mammalian) DNA, and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence. “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g, restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA. Transcriptional and translational controlsequences are “DNA regulatory sequences”, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide for theexpression of a coding sequence in a host cell.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

As used herein, the term “extracellular matrix proteins,” or ECM, refersto four general families of macromolecules, collagens, structuralglycoproteins, proteoglycans and elastins, including fibronectin, andfibrinogen, that provide support and modulate cellular behavior.

“Immunologically effective amounts” are those amounts capable ofstimulating a B cell and/or T cell response.

As used herein, the term “in vivo vaccine” refers to immunization ofanimals with proteins so as to elicit a humoral and cellular responsethat protects against later exposure to the pathogen.

The term “ligand” is used to include molecules, including those withinhost tissues, to which pathogenic bacteria attach.

The term “MHC II antigens” as used herein refers to cell-surfacemolecules that are responsible for rapid graft rejections and arerequired for antigen presentation to T-cells.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen.

The term “oligonucleotide,” as used herein is defined as a moleculecomprised of two or more nucleotides, preferably more than three. Itsexact size will depend upon many factors which, in turn, depend upon theultimate function and use of the oligonucleotide.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an unacceptable allergic or similaruntoward reaction when administered to a human.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be substantially complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a noncomplementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, noncomplementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

A “replicon” is a genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific palindromic nucleotide sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

As used herein, the term “site directed mutagen” refers to a compoundthat can increase the rate at which mutations occur at a certain sitewithin the DNA molecule.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

The term “wound” is used herein to mean the epithelial cellular layer,and other surface structures over tissue, damaged by mechanical,chemical or other influence.

By “immunologically effective amount” is meant an amount of a peptidecomposition that is capable of generating an immune response in therecipient animal. This includes both the generation of an antibodyresponse (B cell response), and/or the stimulation of a cytotoxic immuneresponse (T cell response). The generation of such an immune responsewill have utility in both the production of useful bioreagents, e.g.,CTLs and, more particularly, reactive antibodies, for use in diagnosticembodiments, and will also have utility in various prophylactic ortherapeutic embodiments.

The selected combinations of bacterial binding proteins or fragmentsthereof in the composition used include those binding to fibronectin,fibrinogen, collagen, and elastin. Any such protein, peptide, fragmentthereof, or sequence substantially homologous thereto can be used inthis invention. Illustrative examples are provided below. In addition,bacterial binding proteins or fragments to MHC II analogous

II. Fibronectin-Binding MSCRAMMs

Fibronectin (Fn) is a 440-kDa glycoprotein found in the ECM and bodyfluids of animals. The primary biological function of fibronectinappears to be related to its ability to serve as a substrate for theadhesion of cells expressing the appropriate integrins. Severalbacterial species have been shown to bind fibronectin specifically andto adhere to a fibronectin-containing substratum. Most S. aureusisolates bind Fn, but do so in varying extents, which reflectsvariations in the number of MSCRAMM molecules expressed on the bacterialcell surface. The interaction between Fn and S. aureus is highlyspecific (Kuusela, P., Nature, 276:718-20, 1978). Fn binding is mediatedby two surface exposed proteins with molecular weights of 110 kDa, namedFnBP-A and FnBP-B. The primary Fn binding site consists of a motif of35-40 amino acids, repeated three to five times. The genes for thesehave been cloned and sequenced (Jonsson, K., et al., Eur. J. Biochem.,202:1041-1048, 1991).

WO-A-85/05553 discloses bacterial cell surface proteins havingfibronectin, fibrinogen, collagen, and or laminin binding ability.

U.S. Pat. Nos. 5,320,951 and 5,571,514 to Hook, et al., discloses thefibronectin binding protein A (fnbA) gene sequence, and products andmethods based on this sequence.

U.S. Pat. No. 5,175,096 to Hook et al., discloses the gene sequence offnbB, a hybrid DNA molecule (fnbB) and biological products and methodsbased on this sequence.

U.S. Pat. No. 5,652,217 discloses an isolated and purified proteinhaving binding activity that is encoded by a hybrid DNA molecule from S.aureus of defined sequence.

U.S. Pat. No. 5,440,014 discloses a fibronectin binding peptide withinthe D3 homology unit of a fibronectin binding protein of S. aureus whichcan be used for vaccination of ruminants against mastitis caused bystaphylococcal infections, for treatment of wounds, for blocking proteinreceptors, for immunization of other animals, or for use in a diagnosticassay.

U.S. Pat. No. 5,189,015 discloses a method for the prophylactictreatment of the colonization of a S. aureus bacterial strain having theability to bind to fibronectin in a mammal that includes administeringto the mammal in need of treatment a prophylactically therapeuticallyactive amount of a protein having fibronectin binding properties, toprevent the generation of infections caused by a S. aureus bacterialstrain having the ability to bind fibronectin, wherein the protein has amolecular weight of 87 kDa to 165 kDa.

U.S. Pat. No. 5,416,021 discloses a fibronectin binding protein encodingDNA from Streptococcus dysgalactiae, along with a plasmid that includesDNA encoding for fibronectin binding protein from S. dysgalactiaecontained in E. coli, DNA encoding a fibronectin binding protein from S.dysgalactiae and an E. coli microorganism transformed by DNA encoding afibronectin binding protein from S. dysgalactiae.

It has been observed that antibodies to wild type fibronectin bindingprotein do not substantially inhibit the ability of S. aureus to bind tofibronectin, and thus do not exhibit a significant therapeutic effect,in vivo. PCT/US98/01222 discloses antibodies that block the binding offibronectin to fibronectin binding proteins. The antibodies were raisedagainst a site-directed mutated sequence of fibronectin binding proteinthat does not bind to fibronectin. It was identified that there is arapid complexation of fibronectin with fibronectin binding proteins andfragments in vivo. Peptide epitopes that do not bind to fibronectin,even though based on a fibronectin binding domain of a fibronectinbinding protein, do not form a complex with fibronectin in vivo. Thisallows antibodies to be made against the uncomplexed peptide epitope,which inhibit or block the binding of fibronectin to fibronectin bindingproteins.

III. Collagen-Binding MSCRAMMs

Collagen is the major constituent of cartilage. Collagen (Cn) bindingproteins are commonly expressed by staphylococcal strains. The Cnbinding MSCRAMM of S. aureus adheres to cartilage in a process thatconstitutes an important part of the pathogenic mechanism instaphylococcal infections. (Switalski, et al. Mol. Micro. 7(1), 99-107,1993) Cn binding by S aureus is found to play a role in at least, butnot only, arthritis and septicemia. CNAs with molecular weights of 133,110 and 87 kDa (Patti, J., et al., J. Biol. Chem.,267:4766-4772, 1992)have been identified. Strains expressing CNAs with different molecularweights do not differ in their Cn binding ability (Switalski, L. M., etal, Mol. Microbiol., 7:99-107, 1993).

Staphylococcal strains recovered from the joints of patients diagnosedwith septic arthritis or osteomyelitis almost invariably express a CBP,whereas significantly fewer isolates obtained from wound infectionsexpress this adhesin (Switalski et al., Mol. Microbiol., 7:99-107,1993). Similarly, S. aureus strains isolated from the bones of patientswith osteomyelitis often have an MSCRAMM recognizing the bone-specificprotein, bone sialoprotein (BSP) (Ryden et al., Lancet, 11:515-518,1987). S. aureus colonization of the articular cartilage within thejoint space appears to be an important factor contributing to thedevelopment of septic arthritis.

PCT WO 92/07002 discloses a hybrid DNA molecule which includes anucleotide sequence from S. aureus coding for a protein or polypeptidehaving collagen binding activity and a plasmid or phage comprising thenucleotide sequence. Also disclosed are an E. coli strain expressing thecollagen binding protein, a microorganism transformed by the recombinantDNA, the method for producing a collagen binding protein or polypeptide,and the protein sequence of the collagen binding protein or polypeptide.

The cloning, sequencing, and expression of a gene cna, encoding a S.aureus CBP has been reported (Patti, J., et al., J. Biol. Chem.,267:4766-4772, 1992). The cna gene encodes an 133-kDa adhesin thatcontains structural features characteristic of surface proteins isolatedfrom Gram-positive bacteria.

Recently, the ligand-binding site has been localized within theN-terminal half of the CBP (Patti, J. et al, Biochemistry,32:11428-11435, 1993). By analyzing the Col binding activity ofrecombinant proteins corresponding to different segments of the MSCRAMM,a 168-amino-acid long protein fragment (corresponding to amino acidresidues 151-318) that had appreciable Col binding activity wasidentified. Short truncations of this protein in the N or C terminusresulted in a loss of ligand binding activity but also resulted inconformational changes in the protein as indicated by circular dichroismspectroscopy.

Patti et al. (J of Biol Chem., 270, 12005-12011, 1995) disclose acollagen binding epitope in the S. aureus adhesin encoded by the cnagene. In their study, the authors synthesized peptides derived from thesequence of the said protein and used them to produce antibodies. Someof these antibodies inhibit the binding of the protein to collagen.

PCT/US97/08210 discloses that certain identified epitopes of thecollagen binding protein (M55, M33, and M17) can be used to generateprotective antibodies. The application also discloses the crystalstructure of the CBP which provides critical information necessary foridentifying compositions which interfere with, or block completely, thebinding of Col to CBPS. The ligand-binding site in the S. aureus CBP anda 25-amino-acid peptide was characterized that directly inhibits thebinding of S. aureus to 125 I-labeled type II Col.

IV. Fibrinogen-Binding MSCRAMMs

Fibrin is the major component of blood clots, and fibrinogen/fibrin isone of the major plasma proteins deposited on implanted biomaterials.Considerable evidence exists to suggest that bacterial adherence tofibrinogen/fibrin is important in the initiation of device-relatedinfection. For example, as shown by Vaudaux et al, S. aureus adheres toin vitro plastic that has been coated with fibrinogen in adose-dependent manner (J. Infect. Dis. 160:865-875 (1989)). In addition,in a model that mimics a blood clot or damage to a heart valve, Herrmannet al demonstrated that S. aureus binds avidly via a fibrinogen bridgeto platelets adhering to surfaces (J. Infect. Dis. 167: 312-322 (1993)).S. aureus can adhere directly to fibrinogen in blood clots formed invitro, and can adhere to cultured endothelial cells via fibrinogendeposited from plasma acting as a bridge (Moreillon et al., Infect.Immun. 63:4738-4743 (1995); Cheung et al., J. Clin. Invest. 87:2236-2245(1991)). As shown by Vaudaux et al. and Moreillon et al., mutantsdefective in the fibrinogen-binding protein clumping factor (ClfA)exhibit reduced adherence to fibrinogen in vitro, to explantedcatheters, to blood clots, and to damaged heart valves in the rat modelfor endocarditis (Vaudaux et al., Infect Immun. 63:585-590 (1995);Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

An adhesin for fibrinogen, often referred to as “clumping factor,” islocated on the surface of S. aureus cells. The interaction betweenbacteria and fibrinogen in solution results in the instantaneousclumping of bacterial cells. The binding site on fibrinogen is locatedin the C-terminus of the gamma chain of the dimeric fibrinogenglycoprotein. The affinity is very high and clumping occurs in lowconcentrations of fibrinogen. Scientists have recently shown thatclumping factor also promotes adherence to solid phase fibrinogen, toblood clots, and to damaged heart valves (McDevitt et al., Mol.Microbiol. 11:237-248 (1994); Vaudaux et al., Infect. Immun. 63:585-590(1995); Moreillon et al., Infect. Immun. 63: 4738-4743 (1995)).

Two genes in S. aureus have been found that code for two Fg bindingproteins, ClfA and ClfB. The gene, clfA, was cloned and sequenced andfound to code for a polypeptide of 92 kDa. ClfA binds the gamma chain offibrinogen, and ClfB binds the alpha and beta chains (Eidhin, et al.,Mol Micro, awaiting publication, 1998). ClfB is a cell wall associatedprotein with a predicted molecular weight of 88 kDa and an apparentmolecular weight of 124 kDa that binds both soluble and immobilizedfibrinogen and acts as a clumping factor.

The gene for a clumping factor protein, designated ClfA, was cloned,sequenced and analyzed in detail at the molecular level (McDevitt etal., Mol. Microbiol. 11: 237-248 (1994); McDevitt et al., Mol.Microbiol. 16:895-907 (1995)). The predicted protein is composed of 933amino acids. A signal sequence of 39 residues occurs at the N-terminusfollowed by a 520 residue region (region A), which contains thefibrinogen binding domain. A 308 residue region (region R), composed of154 repeats of the dipeptide serine-aspartate, follows. The R regionsequence is encoded by the 18 basepair repeat GAY TCN GAY TCN GAY AGY(SEQ ID NO: 15) in which Y equals pyrimidines and N equals any base. TheC-terminus of ClfA has features present in many surface proteins ofgram-positive bacteria such as an LPDTG (SEQ ID NO: 16) motif, which isresponsible for anchoring the protein to the cell wall, a membraneanchor, and positive charged residues at the extreme C-terminus.

The platelet integrin alpha IIbβ3 recognizes the C-terminus of the gammachain of fibrinogen. This is a crucial event in the initiation of bloodclotting during coagulation. ClfA and alpha IIbβ3 appear to recognizeprecisely the same sites on fibrinogen gamma chain because ClfA canblock platelet aggregation, and a peptide corresponding to theC-terminus of the gamma chain (198-41 1) can block both the integrin andClfA interacting with fibrinogen (McDevitt et al., Eur. J. Biochem.247:416-424 (1997)). The fibrinogen binding site of alpha IIbβ3 is closeto, or overlaps, a Ca2+ binding determinant referred to as an “EF hand”.ClfA region A carries several EF hand-like motifs. A concentration ofCa2+ in the range of 3-5 mM blocks these ClfA-fibrinogen interactionsand changes the secondary structure of the ClfA protein. Mutationsaffecting the ClfA EF hand reduce or prevent interactions withfibrinogen. Ca2+ and the fibrinogen gamma chain seem to bind to thesame, or to overlapping, sites in ClfA region A.

The alpha chain of the leukocyte integrin, alpha MB2, has an insertionof 200 amino acids (A or I domain) which is responsible for ligandbinding activities. A novel metal ion-dependent adhesion site (MIDAS)motif in the I domain is required for ligand binding. Among the ligandsrecognized is fibrinogen. The binding site on fibrinogen is in the gammachain (residues 190-202). It was recently reported that Candida albicanshas a surface protein, alpha Intlp, having properties reminiscent ofeukaryotic integrins. The surface protein has amino acid sequencehomology with the I domain of MB2, including the MIDAS motif.Furthermore, Intlp binds to fibrinogen.

ClfA region A also exhibits some degree of sequence homology with alphaIntlp. Examination of the ClfA region A sequence has revealed apotential MIDAS motif. Mutations in putative cation coordinatingresidues in the DxSxS portion of the MIDAS motif in ClfA results in asignificant reduction in fibrinogen binding. A peptide corresponding tothe gamma-chain binding site for alpha Mβ2 (190-202) has been shown byO'Connell et al. to inhibit ClfA-fibrinogen interactions (O'Connell, J.Biol. Chem., in press, 1998). Thus it appears that ClfA can bind to thegamma-chain of fibrinogen at two separate sites. The ligand bindingsites on ClfA are similar to those employed by eukaryotic integrins andinvolve divalent cation binding EF-hand and MIDAS motifs.

Also known is the fibrinogen binding protein, ClfB. Used herein are theprotein as well as antibodies to the protein and diagnostic kits thatinclude the protein or its antibodies. ClfB has a predicted molecularweight of approximately 88 kDa and an apparent molecular weight ofapproximately 124 kDa. ClfB is a cell-wall associated protein and bindsboth soluble and immobilized fibrinogen. In addition, ClfB binds boththe alpha and beta chains of fibrinogen and acts as a clumping factor.

Proteins related to the fibrinogen-binding ClfA and ClfB have beenfound, which bind to the extracellular matrix. The SdrC, SdrD and SdrEproteins are related in primary sequence and structural organization tothe ClfA and ClfB proteins, and are also localized on the cell surface.With the A region of these proteins localized on the cell surface, theproteins can interact with the proteins in plasma, the extracellularmatrix or with molecules on the surface of host cells. SdrC can bind tothe extracellular matrix proteins, for example, vitronectin. SdrE alsobinds to the extracellular matrix, for example, SdrE binds bonesialoprotein (BSP).

It has been discovered that in the A region of SdrC, SdrD, ClfA andClfB, there is highly conserved amino acid sequence that can be used toderive a consensus TYTFTDYVD (SEQ ID NO: 17) motif. The motif can beused in multicomponent vaccines to impart broad spectrum immunity tobacterial infections, and also can be used to produce monoclonal orpolyclonal antibodies that impart broad spectrum passive immunity. In analternative embodiment, any combination of the variable sequence motifderived from the Sdr and Clf protein families, (T/I) (Y/F) (T/V) (F) (T)(D/N) (Y) (V) (D/N), can be used to impart immunity or to induceprotective antibodies.

V. Elastin-Binding MSCRAMMs

The primary role of elastin is to confer the property of reversibleelasticity to tissues and organs (Rosenbloom, J., et al., FASEB J.,7:1208-1218, 1993). Elastin expression is highest in the lung, skin andblood vessels, but the protein is widely expressed in mammalian hostsfor S. aureus. S. aureus binding to elastin was found to be rapid,reversible, of high affinity and ligand specific. Furthermore, a 25 kDacell surface elastin binding protein (EbpS) was isolated and proposed tomediate S. aureus binding to elastin-rich host ECM. EbpS binds to aregion in the N-terminal 30 kDa fragment of elastin.

PCT/US97/03106 discloses the gene sequences for an elastin bindingprotein. DNA sequence data disclosed indicates that the ebps openreading frame consists of 606 bp, and encodes a novel polypeptide of 202amino acids. EbpS protein has a predicted molecular mass of 23,345daltons and pI of 4.9. EbpS was expressed in E. coli as a fusion proteinwith polyhistidine residues attached to the N-terminus. A polyclonalantibody raised against recombinant EbpS interacted specifically withthe 25 kDa cell surface EbpS and inhibited staphylococcal elastinbinding. Furthermore, recombinant EbpS bound specifically to immobilizedelastin and inhibited binding of Staphylococcus aureus to elastin. Adegradation product of recombinant EbpS lacking the first 59 amino acidsof the molecule and a C-terminal fragment of CNBr-cleaved recombinantEbpS, however, did not interact with elastin. These results stronglysuggest that EbpS is the cell surface molecule mediating binding ofStaphylococcus aureus to elastin. The finding that some constructs ofrecombinant EbpS do not interact with elastin suggests that the elastinbinding site in EbpS is contained in the first 59 amino acids of themolecule.

Several independent criteria indicate that EbpS is the surface proteinmediating cellular elastin binding. First, rEbpS binds specifically toimmobilized elastin and inhibits binding of S. aureus cells to elastinin a dose dependent manner. These results establish that EbpS is anelastin binding protein that is functionally active in a soluble form.Second, an antibody raised against rEbpS recognizes a 25 kDa proteinexpressed on the cell surface of S. aureus cells. In addition to thesize similarity and antibody reactivity, further evidence that this 25kDa protein is cell surface EbpS is provided by the experiment showingthat binding of the 25 kDa protein to immobilized anti-rEbpS IgG isinhibited in the presence of excess unlabeled rEbpS. Finally, Fabfragments prepared from the anti-rEbpS antibody, but not from itspre-immune control, inhibit binding of S. aureus to elastin. This resultsuggests that the topology of surface EbpS is such that the elastinbinding site is accessible to interact with ligands (i.e. elastin andthe anti-rEbpS Fab fragment) and not embedded in the cell wall ormembrane domains. The composite data demonstrate that EbpS is the cellsurface protein responsible for binding S. aureus to elastin.

The present and previous findings suggest the existence of afunctionally active 40 kDa intracellular precursor form of EbpS thatrequires processing at the C terminus prior to surface expression. Thisnotion is based on the following observations: i) there exists anintracellular 40 kDa elastin binding protein that is never detectedduring cell surface labeling experiments, ii) the 25 kDa EbpS and the 40kDa elastin binding protein have an identical N-terminal sequence, andiii) a single gene exists for EbpS. Because the size of the ebps openreading frame is not sufficient to encode a 40 kDa protein, at first theinventors disregarded this hypothesis. However, their studies with rEbpSdemonstrated that although the actual size of the recombinant protein is26 kDa, it migrates aberrantly as a 45 kDa protein in SDS-30 PAGE. Thisfinding suggests that full length native EbpS, with a predicted size of23 kDa, may be migrating in SDS-PAGE as the 40 kDa intracellularprecursor, and that the 25 kDa surface form of EbpS is actually asmaller form of the molecule processed at the C-terminus. Although EbpSlacks an N-terminal signal peptide and other known sorting and anchoringsignals, this proposed intracellular processing event may explain somequestions regarding how EbpS is targeted to the cell surface. In fact,C-terminal signal peptides have been identified in several bacterialproteins (Fath, M. J. and Kolter, R., Microbiol. Rev., 57:995-1017,1993) and alternative means of anchoring proteins to the cells surfacehave been reported in gram positive bacteria (Yother, J. and White, J.M., J. Bacteriol., 176:2976-2985, 1994).

Using overlapping EbpS fragments and recombinant constructs, the elastinbinding site in EbpS was mapped to the amino terminal domain of themolecule (PCT/US97/03106). Overlapping synthetic peptides spanning aminoacids 14-34 were then used to better define the binding domain. Amongthese, peptides corresponding to residues 14-23 and 18-34 specificallyinhibited elastin binding by more than 95%. Common to all activesynthetic peptides and proteolytic and recombinant fragments of EbpS isthe hexameric sequence ¹⁸Thr-Asn-Ser-His-Gln-Asp²³ (SEQ ID NO: 18).Further evidence that this sequence is important for elastin binding wasthe loss of activity when Asp²³ was substituted with Asn in thesynthetic peptide corresponding to residues 18-34. However, thesynthetic hexamer TNSHQD (SEQ ID NO: 18) by itself did not inhibitstaphylococcal binding to elastin. These findings indicate that althoughthe presence of the TNSHQD sequence is essential for EbpS activity,flanking amino acids in the N- or C-terminal direction and the carboxylside chain of Asp²³ are required for elastin recognition.

VI. MHC II-Analogous Proteins, (MAP)

In addition to fibrinogen, fibronectin, collagen and elastin, S. aureusstrains associate with other adhesive eukaryotic proteins, many of whichbelong to the family of adhesive matrix proteins, such as vitronectin.(Chatwal et al., Infect. Immun., 55:1878-1883, 1987). U.S. Pat. No.5,648,240 discloses a DNA segment comprising a gene encoding a S. aureusbroad spectrum adhesin that has a molecular weight of about 70 kDa. Theadhesin is capable of binding fibronectin or vitronectin and includes aMHC II mimicking unit of about 30 amino acids. Further analyses of thebinding specificities of this protein reveal that it functionallyresembles an MHC II antigen in that it binds synthetic peptides. Thus,in addition to mediating bacterial adhesion to ECM proteins, it may playa role in staphylococcal infections by suppressing the immune system ofthe host. The patent further claims a recombinant vector that includesthe specified DNA sequence, a recombinant host cell transformed with thevector, and DNA which hybridizes with the DNA of specified sequence.Also disclosed is a composition that includes a protein or polypeptideencoded by the specified DNA sequence and a method of inducing an immuneresponse in an animal that includes administering an immunogeniccomposition that includes the encoded protein or polypeptide. A methodof making a MHC II antigen protein analog comprising the steps ofinserting the specified DNA sequence in a suitable expression vector andculturing a host cell transformed with the vector under conditions toproduce the MHC II antigen protein analog is additionally claimed in thepatent.

VII. SDR Proteins from Staphylococcus Epidermidis

Staphylococcus epidermidis, a coagulase-negative bacterium, is a commoninhabitant of human skin and a frequent cause of foreign-bodyinfections. Pathogenesis is facilitated by the ability of the organismto first adhere to, and subsequently to form biofilms on, indwellingmedical devices such as artificial valves, orthopedic devices, andintravenous and peritoneal dialysis catheters. Device-related infectionsmay jeopardize the success of medical treatment and significantlyincrease patient mortality. Accordingly, the ability to develop vaccinesthat can control or prevent outbreaks of S. epidermidis infection is ofgreat importance, as is the development of multicomponent vaccines thatcan prevent or treat infection from a broad spectrum of bacteria,including both coagulase-positive and coagulase negative bacteria at thesame time.

Three Sdr (serine-aspartate (SD) repeat region) proteins that areexpressed by S. epidermidis have been designated as SdrF, SdrG and SdrH,and the amino acid sequences of these proteins and their nucleic acidsequences are shown in FIGS. 3-5, respectively. In addition, a morecomplete description of these proteins is provided in a co-pending U.S.patent application of Foster et al. which is based on U.S. provisionalapplication Ser. Nos. 60/098,443 and 60/117,119. These applications areincorporated herein by reference.

In accordance with the present invention, a composition useful as avaccine is provided that includes the components of any of the aboveembodiments in combination with an SdrF, SdrG or an SdrH protein. Inaddition, antibodies to these proteins can be raised using conventionalmeans, and antibodies to the SdrF, SdrG or an SdrH proteins can beemployed in any of the above combinations which employ antibodies to theother adhesins discussed herein. The compositions and vaccines whichinclude an SDR protein such as SdrF, SdrG or SdrH can thus be used totreat a broad spectrum of bacterial infections, including those arisingboth from coagulase-positive and coagulase-negative bacteria.

VIII. Bacterial Components

In an embodiment of the invention, a composition is provided thatincludes the components of any of the above embodiments in combinationwith a bacterial component, preferably capsular polysaccharides type 5or type 8, to increase the rate of opsonization and phagocytosis of S.aureus.

Staphylococci contain antigenic polysaccharides, such as capsularpolysaccharide types 5 and 8, and proteins as well as other substancesimportant in cell wall structure. Peptidoglycan, a polysaccharidepolymer containing linked subunits, provides the rigid exoskeleton ofthe cell wall. Peptidoglycan is destroyed by strong acids or exposure tolysozyme. It is important in the pathogenesis of infection. It elicitsproduction of interleukin-1 (endogenous pyrogen) and opsonic antibodiesby monocytes. It can be a chemoattractant for polymorphonuclearleukocytes, have endotoxin-like activity, produce a localized Shwartzmanphenomenon, and activate complement.

Teichoic acids, lipoteichoic acid for example, which are polymers ofglycerol or ribotol phosphate, are linked to the peptidolglycan and canbe antigenic. Antiteichoic antibodies detectable by gel diffusion may befound in patients with active endocarditis due to S. aureus.

Protein A is a cell wall component of many S. aureus strains that bindsto the Fc portion of IgG molecules except IgG3. The Fab portion of IgGbound to protein A is free to combine with a specific antigen. Protein Ahas become an important reagent in immunology and diagnostic laboratorytechnology; for example, protein A with attached IgG molecules directedagainst a specific bacterial antigen will agglutinate bacteria that havethat antigen (“coagglutination”).

Some S. aureus strains have capsules, which inhibit phagocytosis bypolymorphonuclear leukocytes unless specific antibodies are present.Most strains of S. aureus have coagulase, or clumping factor, on thecell wall surface; coagulase binds nonenzymatically to fibrinogen,yielding aggregation of the bacteria.

Staphylococci can produce disease both through their ability to multiplyand spread widely in tissues and through their production of manyextracellular substances. Some of these substances are enzymes; othersare considered to be toxins, though they may function as enzymes. Manyof the toxins are under the genetic control of plasmids; some may beunder both chromosomal and extrachromosomal control; and for others themechanism of genetic control is not well defined.

A. Catalase:

Staphylococci produce catalase, which converts hydrogen peroxide intowater and oxygen. The catalase test differentiates the staphylococci,which are positive, from the streptococci, which are negative.

B. Coagulase:

S. aureus produces coagulase, an enzyme-like protein that clots oxalatedor citrated plasma in the presence of a factor contained in many sera.The serum factor reacts with coagulase to generate both esterase andclotting activities, in a manner similar to the activation ofprothrombin to thrombin. The action of coagulase circumvents the normalplasma clotting cascade. Coagulase may deposit fibrin on the surface ofstaphylococci, perhaps altering their ingestion by phagocytic cells ortheir destruction within such cells. Coagulase production is consideredsynonymous with invasive pathogenic potential. However,coagulase-negative bacteria such as S. epidermidis also pose a threatfor serious infection as well.

C. Other Enzymes:

Other enzymes produced by staphylococci include a hyaluronidase, orspreading factor; a staphylokinase resulting in fibrinolysis but actingmuch more slowly than streptokinase; proteinases; lipases; andβ-lactamase.

D. Exotoxins:

These include several toxins that are lethal for animals on injection,cause necrosis in skin, and contain soluble hemolysins which can beseparated by electrophoresis. The alpha toxin (hemolysin) is aheterogeneous protein that can lyse erythrocytes and damage plateletsand is probably identical with the lethal and dermonecrotic factors ofexotoxin. Alpha toxin also has a powerful action on vascular smoothmuscle. Beta toxin degrades sphingomyelin and is toxic for many kinds ofcells, including human red blood cells. These toxins and two others, thegamma and delta toxins; are antigenically distinct and bear norelationship to streptococcal lysins. Exotoxin treated with formalingives a non-poisonous but antigenic toxoid, but this is not clinicallyuseful.

E. Leukocidin:

This toxin of S. aureus can kill exposed white blood cells of manyanimals. Its role in pathogenic staphylococci may not kill white bloodcells and may be phagocytosed as effectively as nonpathogenic varieties.However, they are capable of very active intra-cellular multiplication,whereas the nonpathogenic organisms tend to die inside the cell.Antibodies to leukocidin may plan a role in resistance to recurrentstaphylococcal infections.

F. Exfoliative Toxin:

This toxin of S. aureus includes at least two proteins that yield thegeneralized desquamation of the staphylococcal scaled skin syndrome.Specific antibodies protect against the exfoliative action of the toxin.

G. Toxic Shock Syndrome Toxin.

Most S. aureus strains isolated from patients with toxic shock syndromeproduce a toxin called toxic shock syndrome toxin-1 (TSST-1), which isthe same as enterotoxin F and pyrogenic exotoxin C. TSST-1 is theprototypical superantigen which promotes the protean manifestations ofthe toxic shock syndrome. In humans, the toxin is associated with fever,shock, and multisystem involvement, including a desquamative skin rash.In rabbits, TSST-1 produces fever, enhanced susceptibility to theeffects of bacterial lipopolysaccharides, and other biologic effectssimilar to toxic shock syndrome, but the skin rash and desquamation donot occur.

H. Enterotoxins:

There are at least six (A-F) soluble toxins produced by nearly 50% of Saureus strains. Like TSST-1, the enterotoxins are superantigens thatbind to MHC class II molecules, yielding T cell stimulation. Theenterotoxins are heat-stable (they resist boiling for 30 minutes) andare resistant to the action of gut enzymes. An important cause of foodpoisoning, enterotoxins are produced when S. aureus grows incarbohydrate and protein foods. The gene for enterotoxin production maybe on the chromosome, but a plasmid may carry a protein that regulatesactive toxin production. Ingestion of 25 μg of enterotoxin B by humansor monkeys results in vomiting and diarrhea. The emetic effect ofenterotoxin is probably the result of central nervous system stimulation(vomiting center) after the toxin acts on neural receptors in the gut.Enterotoxins can be assayed by precipitin tests (gel diffusion).

There are also many other antigenic proteins produced by Staphylococcalorganisms. These include the MSCRAMMs mentioned above, as well as: bonesialoprotein binding protein, clusterin binding protein, heparin sulfatebinding protein, thrombospondin binding protein, transferrin bindingprotein and vitronectin binding protein. S. aureus further expressesvirulence factors such as phophatidyl phospholipase, and toxinexpression regulators such as Rap protein.

IX. Proteins and Peptides with Substantial Homology or EquivalentFunction to Those Described Herein

The disclosed compositions can include, as desired, full sequenceproteins, peptides, protein or peptide fragments, isolated epitopes,fusion proteins, or any alternative which binds to the target ECM,whether in the form of a wild type, a site-directed mutant, or asequence which is substantially homologous thereto.

Two DNA sequences are “substantially homologous” when at least about70%, (preferably at least about 80%, and most preferably at least about90 or 95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual,1982; DNA Cloning, Vols. I & II supra; Nucleic Acid Hybridization, [B.D. Hames & S. J. Higgins eds. (1985)].

When used in conjunction with amino acid sequences, the term“substantially similar” means an amino acid sequence which is notidentical to published sequences, but which produces a protein havingthe same functionality and activities, either because one amino acid isreplaced with another similar amino acid, or because the change (whetherit be substitution, deletion or insertion) does not substantially effectthe active site of the protein. Two amino acid sequences are“substantially homologous” when at least about 70%, (preferably at leastabout 80%, and most preferably at least about 90% or 95%) of the aminoacids match over the defined length of the sequences.

It should also be understood that each of the MSCRAMM polypeptides ofthis invention may be part of a larger protein. For example, a ClfApolypeptide of this invention may be fused at its N-terminus orC-terminus to a ClfB polypeptide, or to a non-fibrinogen bindingpolypeptide or combinations thereof. Polypeptides which may be usefulfor this purpose include polypeptides derived any of the MSCRAMMproteins, and serotypic variants of any of the above. Non-MSCRAMMpolypeptides which may be useful for this purpose include any of thebacterial components described above.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according toTable 1. It should be understood by one skilled in the art that thecodons specified in Table 1 are for RNA sequences. The correspondingcodons for DNA have a T substituted for U. In keeping with standardnomenclature (J. Biol. Chem., 243:3552-3559, 1969), abbreviations foramino acid residues are further shown in Table I.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

TABLE I Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GCG GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG GUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, J Mol Biol, 157(1):105-132,1982, incorporate herein by reference). It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics (Kyte and Doolittle, supra1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+1.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

The polypeptides of the present invention can be can be chemicallysynthesized. The synthetic polypeptides are prepared using the wellknown techniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (N^(a)-amino protected N^(a)-t-butyloxycarbonyl) amino acidresin with the standard deprotecting, neutralization, coupling and washprotocols of the original solid phase procedure of Merrifield [J. Am.Chem. Soc., 85:2149-2154 (1963)], or the base-labile N^(a)-aminoprotected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first describedby Carpino and Han [J. Org. Chem., 37:3403-3409 (1972)]. Both Fmoc andBoc N^(a)-amino protected amino acids can be obtained from Fluka,Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical,Bachem, or Peninsula Labs or other chemical companies familiar to thosewho practice this art. In addition, the method of the invention can beused with other N^(a)-protecting groups that are familiar to thoseskilled in this art. Solid phase peptide synthesis may be accomplishedby techniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford, Ill.; Fields et al., Int. J. Pept. Protein es.35:161-214 (1990), or using automated synthesizers, such as sold by ABS.Thus, polypeptides of the invention may comprise D-amino acids, acombination of D- and L-amino acids, and various “designer” amino acids(e.g., β-methyl amino acids, Ca-methyl amino acids, and Na-methyl aminoacids, etc.) to convey special properties. Synthetic amino acids includeornithine for lysine, fluoro-phenylalanine for phenylalanine, andnorleucine for leucine or isoleucine. Additionally, by assigningspecific amino acids at specific coupling steps, a-helices, β turns, βsheets, β-turns, and cyclic peptides can be generated.

In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids will be resistant to L-amino acid-specificproteases in vivo. In addition, the present invention envisionspreparing peptides that have more well defined structural properties,and the use of peptidomimetics, and peptidomimetic bonds, such as esterbonds, to prepare peptides with novel properties. In another embodiment,a peptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a molecule would be resistant to peptide bond hydrolysis,e.g., protease activity. Such peptides would provide ligands with uniquefunction and activity, such as extended half-lives in vivo due toresistance to metabolic breakdown, or protease activity. Furthermore, itis well known that in certain systems constrained peptides show enhancedfunctional activity (Hruby, Life Sciences, 31:189-199 (1982)); (Hruby etal, Biochem J. 268:249-262 (1990)].

The following non-classical amino acids may be incorporated in thepeptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., J. Am.Chem. Soc., 113:2275-2283, 1991); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett.,1991); 2-aminotetrahydro-naphthalene-2-carboxylic acid (Landis, Ph.D.Thesis, University of Arizona, 1989);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al, J.Takeda Res. Labs., 43:53-76, 1989); β-carboline (D and L) (Kazmierski,Ph.D. Thesis, University of Arizona, 1988); HIC (histidine isoquinolinecarboxylic acid) (Zechel et al, Int. J. Pep. Protein Res., 43, 1991);and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al., J. Org. Chem., 50:5834-5838 (1985)];β-sheet inducing analogs [Kemp et al., Tetrahedron Lett., 29:5081-5082(1988)]; β-turn inducing analogs (Kemp et al., Tetrahedron Lett.,29:5057-5060 (1988)]; alpha-helix inducing analogs [Kemp et al.,Tetrahedron Lett., 29:4935-4938 (1988)]; β-turn inducing analogs [Kempet al., J. Org. Chem., 54:109:115 (1989)]; and analogs provided by thefollowing references: Nagai and Sato, Tetrahedron Lett., 26:647-650(1985); DiMaio et al., J. Chem. Soc. Perkin Trans., p. 1687 (1989); alsoa Gly-Ala turn analog (Kahn et al., Tetrahedron Lett., 30:2317, 1989);amide bond isostere (Jones et al., Tetrahedron Lett., 29:3853-3856,1989); tetrazole (Zabrocki et al., J. Am. Chem. Soc., 110:5875-5880,1988); DTC (Samanen et al., Int. J. Protein Pep. Res., 35:501:509,1990); and analogs taught in Olson et al., (J. Am. Chem. Sci.,112:323-333, 1990) and Garvey et al.,(J. Org. Chem., 56:436, 1990).Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

X. Uses for MSCRAMM and Antibody Compositions

The protein compositions disclosed herein can be used for the treatmentof wounds, for blocking protein receptors or for immunization(vaccination). In the latter case, the body creates specific antibodies,which can protect against invasion by bacterial strains comprising sucha cell surface protein, and whereby the antibodies block the adherenceof the bacterial strains to a damaged tissue.

The protein composition can be dispersed in a sterile, isotonic salinesolution, optionally with the addition of a pharmaceutically acceptabledispersing agent. Different types of adjuvants can further be used tosustain the release in the tissue, and thus expose the peptide for alonger time to the immune defense system of a body.

The proteins, nucleic acid molecules or antibodies are useful forinterfering with the initial physical interaction between a pathogen andmammalian host responsible for infection, such as the adhesion ofbacteria, particularly gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block protein-mediated mammalian cellinvasion; to block bacterial adhesion between mammalian extracellularmatrix proteins and bacterial proteins that mediate tissue damage; and,to block the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or surgicaltechniques. Medical devices or polymeric biomaterials to be coated withthe antibodies, proteins and active fragments described herein include,but are not limited to, staples, sutures, replacement heart valves,cardiac assist devices, hard and soft contact lenses, intraocular lensimplants (anterior chamber, posterior chamber or phasic), other implantssuch as corneal inlays, kerato-prostheses, vascular stents,epikeratophalia devices, glaucoma shunts, retinal staples, scleralbuckles, dental prostheses, thyroplastic devices, laryngoplasticdevices, vascular grafts, soft and hard tissue prostheses including, butnot limited to, pumps, electrical devices including stimulators andrecorders, auditory prostheses, pacemakers, artificial larynx, dentalimplants, mammary implants, penile implants, cranio/facial tendons,artificial joints, tendons, ligaments, menisci, and disks, artificialbones, artificial organs including artificial pancreas, artificialhearts, artificial limbs, and heart valves; stents, wires, guide wires,intravenous and central venous catheters, laser and balloon angioplastydevices, vascular and heart devices (tubes, catheters, balloons),ventricular assists, blood dialysis components, blood oxygenators,urethra/ureteral/urinary devices (Foley catheters, stents, tubes andballoons), airway catheters (endotracheal and tracheostomy tubes andcuffs), enteral feeding tubes (including nasogastric, intragastric andjejunal tubes), wound drainage tubes, tubes used to drain the bodycavities such as the pleural, peritoneal, cranial, and pericardialcavities, blood bags, test tubes, blood collection tubes, vacutainers,syringes, needles, pipettes, pipette tips, and blood tubing.

The term “coated” or “coating”, as used herein, means to apply theprotein, antibody, or active fragment to a surface of the device,preferably an outer surface that would be exposed to S. aureusinfection. The surface of the device need not be entirely covered by theprotein, antibody or active fragment.

XI. Preparation of Proteins DNA, and Antibodies

The skilled reader can employ conventional molecular biology,microbiology, and recombinant DNA techniques to prepare the proteins,peptides, and antibody compositions described herein. Such techniquesare explained fully in the literature. See, e.g., Sambrook et al,Molecular Cloning: A Laboratory Manual (1989); Current Protocols inMolecular Biology Volumes I-III (Ausubel, R. @-I ed., 1994); CellBiology: A Laboratory Handbook Volumes I-III (J. E. Celis, ed., 1994);Current Protocols in Immunology Volumes I-III (Coligan, J. E., ed.,1994); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds., 1985); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins, eds., 1984); Animal CellCulture [R. I. Freshney, ed. 1, (1986); Immobilized Cells And Enzymes[IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning(1984).

Reference to antibodies throughout the specification includes wholepolyclonal and monoclonal antibodies, and parts thereof, either alone orconjugated with other moieties. Antibody parts include Fab and F(ab)2fragments and single chain antibodies. The antibodies may be made invivo in suitable laboratory animals or in vitro using recombinant DNAtechniques. An antibody can be a polygonal or a monoclonal antibody. Ina preferred embodiment, an antibody is a polyclonal antibody. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., Harlow and Lane, Antibodies: a Laboratory Manual, Cold SpringHarbor, N.Y., 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Antibodies, both polyclonal and monoclonal, specific for MSCRAMMepitopes may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of particular binding MSCRAMMs (eithersynthetic peptides, site-specifically mutated, or truncated peptides)can be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst epitope-containing MSCRAMM peptides.

Polyclonal antisera may be obtained, after allowing time for antibodygeneration, simply by bleeding the animal and preparing serum samplesfrom the whole blood.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen, as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, alsomay be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

One of the important features provided by the present invention is apolygonal sera that is relatively homogenous with respect to thespecificity of the antibodies therein. Typically, polygonal antisera isderived from a variety of different “clones,” i.e., B-cells of differentlineage. Monoclonal antibodies, by contrast, are defined as coming fromantibody-producing cells with a common B-cell ancestor, hence their“mono” clonality.

When peptides are used as antigens to raise polyclonal sera, one expectsconsiderably less variation in the clonal nature of the sera than if awhole antigen were employed. Unfortunately, if incomplete fragments ofan epitope are presented, the peptide may very well assume multiple (andprobably non-native) conformations. As a result, even short peptides canproduce polyclonal antisera with relatively plural specificities and,unfortunately, an antisera that does not react or reacts poorly with thenative molecule.

Polyclonal antisera according to the present invention is producedagainst peptides that are predicted to comprise whole, intact epitopes.It is believed that these epitopes are, therefore, more stable in animmunologic sense and thus express a more consistent immunologic targetfor the immune system. Under this model, the number of potential B-cellclones that will respond to this peptide is considerably smaller and,hence, the homogeneity of the resulting sera will be higher. In variousembodiments, the present invention provides for polyclonal antiserawhere the clonality, i.e., the percentage of clone reacting with thesame molecular determinant, is at least 80%. Even higher clonality—90%,95% or greater—is contemplated.

To obtain monoclonal antibodies, one also initially immunizes anexperimental animal, often preferably a mouse, with an MSCRAMM-derivedepitope-containing composition. One then, after a period of timesufficient to allow antibody generation, obtains a population of spleenor lymph cells from the animal. The spleen or lymph cells are then fusedwith cell lines, such as human or mouse myeloma strains, to produceantibody-secreting hybridomas. These hybridomas may be isolated toobtain individual clones which can then be screened for production ofantibody to the desired peptide. Following immunization, spleen cellsare removed and fused, using a standard fusion protocol withplasmacytoma cells to produce hybridomas secreting monoclonal antibodiesagainst MSCRAMM-derived epitopes. Hybridomas which produce monoclonalantibodies to the selected antigens are identified using standardtechniques, such as ELISA and Western blot methods. Hybridoma clones canthen be cultured in liquid media and the culture supernatants unified toprovide the MSCRAMM-derived epitope-specific monoclonal antibodies.

Immortal antibody-producing cell lines can also be created by techniquesother than fusion, such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M.Schreier et al., Hybridoma Techniques (1980); Hammerling et al.,Monoclonal Antibodies And T-cell Hybridomas (1981); Kennett et al.,Monoclonal Antibodies (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods, as well as other procedures which mayutilize antibody specific to the MSCRAMM epitopes.

Additionally, it is proposed that monoclonal antibodies specific to theparticular MSCRAMM-derived peptides may be utilized in other usefulapplications. For example, their use in immunoabsorbent protocols may beuseful in purifying native or recombinant peptide species or syntheticor natural variants thereof.

In general, both poly- and monoclonal antibodies against these peptidesmay be used in a variety of embodiments. For example, they may beemployed in antibody cloning protocols to obtain cDNAs or genes encodingthe peptides discussed herein or related proteins. They may also be usedin inhibition studies to analyze the effects of MSCRAMM-derived peptidesin cells or animals. Anti-MSCRAMM epitope antibodies will also be usefulin immunolocalization studies to analyze the distribution of MSCRAMMsduring various cellular events, for example, to determine the cellularor tissue-specific distribution of the MSCRAMM peptides under differentphysiological conditions. A particularly useful application of suchantibodies is in purifying native or recombinant MSCRAMMs, for example,using an antibody affinity column. The operation of all suchimmunological techniques will be known to those of skill in the art inlight of the present disclosure.

Techniques for the production of single chain antibodies are known tothose skilled in the art and described in U.S. Pat. No. 4,946,778 andcan be used to produce single chain antibodies to the proteins describedherein. Phage display technology may be used to select antibody geneshaving binding, activities for MSCRAMMs, or antigenic portions thereof,from PCR-amplified v genes of lymphocytes from humans screened forhaving antibodies to MSCRAMMs or naive libraries. Bispecific antibodieshave two antigen binding domains wherein each domain is directed againsta different epitope.

The antibody may be labeled directly with a detectable label foridentification and quantification of a staphylococcal bacterium such asS. aureus. Labels for use in immunoassays are generally known to thoseskilled in the art and include enzymes, radioisotopes, and fluorescent,luminescent and chromogenic substances including colored particles suchas colloidal gold and latex beads. Suitable immunoassays includeenzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody may be labeled indirectly by reaction withlabeled substances that have an affinity for immunoglobulin, such asprotein A or G or second antibodies. The antibody may be conjugated witha second substance and detected with a labeled third substance having anaffinity for the second substance conjugated to the antibody. Forexample, the antibody may be conjugated to biotin and theantibody-biotin conjugate detected using labeled avidin or streptavidin.Similarly, the antibody may be conjugated to a hapten and theantibody-hapten conjugate detected using labeled anti-hapten antibody.These and other methods of labeling antibodies and assay conjugates arewell known to those skilled in the art. Antibodies to the bindingproteins may also be used in production facilities or laboratories toisolate additional quantities of the protein, such as by affinitychromatography.

In general, the preparation of bispecific antibodies is also well knownin the art, as exemplified by Glennie et al. (J Immunol, 139:2367-2375,1987). Bispecific antibodies have been employed clinically, for example,to treat cancer patients (Bauer et al, Vox Sang, 61:156-157, 1991). Onemethod for the preparation of bispecific antibodies involves theseparate preparation of antibodies having specificity for differentepitopes of one or more fibronectin binding domains from one or morefibronectin binding protein(s).

While numerous methods are known in the art for the preparation ofbispecific antibodies, the Glennie et al., (1987 supra) method involvesthe preparation of peptic F(ab′Y)2 fragments from the two chosenantibodies, followed by reduction of each to provide separate Fab′YSHfragments. The SH groups on one of the two partners to be coupled arethen alkylated with a cross-linking reagent such aso-phenylenedimaleimide to provide free maleimide groups on one partner.This partner may then be conjugated to the other by means of a thioetherlinkage, to give the desired F(ab′Y)2 heterdconjugate.

Due to ease of preparation, high yield and reproducibility, the Glennieet al., (1987 supra) method is often preferred for the preparation ofbispecific antibodies, however, there are numerous other approaches thatcan be employed and that are envisioned by the inventors. For example,other techniques are known wherein cross-linking with SPDP or protein Ais carried out, or a specific construct is prepared (Titus et al, J.Immunol., 138:4018-4022, 1987; Tutt et al, Eur J Immunol, 21:1351-1358,1991).

Another method for producing bispecific antibodies is by the fusion oftwo hybridomas to form a quadroma (Flavell et al, Br. J. Cancer,64(2):274-280, 1991; Pimm et al, J. Cancer Res Clin Oncol, 118:367-370,1992; French et al, Cancer Res, 51:2358-2361, 1991; Embleton et al., Br.J. Cancer, 63(5):670-674, 1991). As used herein, the term “quadroma” isused to describe the productive fusion of two B cell hybridomas. Usingnow standard techniques, two antibody producing hybridomas are fused togive daughter cells, and those cells that have maintained the expressionof both sets of clonotype immunoglobulin genes are then selected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use if an alternativeassay for the isolation of a preferred quadroma.

In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen mAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁷ M to 1×10⁻⁵M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the second desired MAbis used to generate the quadromas by standard cell fusion techniques(Galfre et al, Methods Enzymol, 73:1-46, 1981), or by using the protocoldescribed by Clark et al (Int J Cancer, 2:15-17, 1988). Briefly, 4.5×10⁷HAT-sensitive first cells are mixed with 2.8×phosphate buffered saline)for 30 in minutes on ice before fusion. Cell fusion is induced usingpolyethylene glycol (PEG) and the cells are plated out in 96 wellmicroculture plates. Quadromas are selected using Hat-containing medium.Bispecific antibody-containing cultures are identified using, forexample, a solid phase isotype-specific ELISA and isotype-specificimmunofluorescence staining.

In one identification embodiment to identify the bispecific antibody,the wells of microliter plates (Falcon, Becton Dickinson Labware) arecoated with a reagent that specifically interacts with one of the parenthybridoma antibodies and that lacks cross-reactivity with bothantibodies. The plates are washed, blocked, and the supernatants (SNs)to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., p-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD410 values determined using an ELISA reader.

In another identification embodiment, microliter plates pre-treated withpoly-L-lysine are used to bind one of the target cells to each well, thecells are then fixed, e.g. using 1% glutaraldehyde, and the bispecificantibodies are tested for their ability to bind to the intact cell. Inaddition, FACS, immunofluorescence staining, idiotype specificantibodies, antigen binding competition assays, and other methods commonin the art of antibody characterization may be used in conjunction withthe present invention to identify preferred quadromas.

Following the isolation of the quadroma, the bispecific antibodies arepurified away from other cell products. This may be accomplished by avariety of protein isolation Procedures, known to those skilled in theart of immunoglobulin purification. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g.,Antibodies: A Laboratory Manual, 1988).

For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

Purified BsAbs and parental antibodies may also be characterized andisolated by SDS PAGE electrophoresis, followed by staining with silveror Coomassie. This is possible when one of the parental antibodies has ahigher molecular weight than the other, wherein the band of the BsAbsmigrates midway between that of the two parental antibodies. Reductionof the samples verifies the presence of heavy chains with two differentapparent molecular weights.

Furthermore, recombinant technology is now available for the preparationof antibodies in general, allowing the preparation of recombinantantibody genes encoding an antibody having the desired dual specificity(Van Duk et al., Int J. Cancer, 43:344-349, 1989). Thus, after selectingthe monoclonal antibodies having the most preferred bindingcharacteristics, the respective genes for these antibodies can beisolated, e.g., by immunological screening of a phage expression library(Oi and Morrison, 1986; Winter and Milstein, 1991). Then, throughrearrangement of Fab coding domains, the appropriate chimeric constructcan be readily obtained.

Humanized monoclonal antibodies are antibodies of animal origin thathave been modified using genetic engineering techniques to replaceconstant region and/or variable region framework sequences with humansequences, while retaining the original antigen specificity.

Such antibodies are commonly derived from rodent antibodies withspecificity against human antigens. Such antibodies are generally usefulfor in vivo therapeutic applications. This strategy reduces the hostresponse to the foreign antibody and allows selection of the humaneffector functions.

The techniques for producing humanized immunoglobulins are well known tothose of skill in the art. For example U.S. Pat. No. 5,693,762 disclosesmethods for producing, and compositions of, humanized immunoglobulinshaving one or more complementarily determining regions (CDR's). Whencombined into an intact antibody, the humanized immunoglobulins aresubstantially non-immunogenic in humans and retain substantially thesame affinity as the donor immunoglobulin to the antigen, such as aprotein or other compound containing an epitope.

Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present invention includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobin preparations and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

U.S. Pat. No. 5,565,332 describes methods for the production ofantibodies, or antibody fragments, which have the same bindingspecificity as a parent antibody but which have increased humancharacteristics. Humanized antibodies may be obtained by chainshuffling, perhaps using phage display technology, in as much as suchmethods will be useful in the present invention the entire text of U.S.Pat. No. 5,565,332 is incorporated herein by reference.

Using the peptide antigens described herein, the present invention alsoprovides methods of generating an immune response, which methodsgenerally comprise administering to an animal, apharmaceutically-acceptable composition comprising an immunologicallyeffective amount of an MSCRAMM-derived peptide composition. Preferredanimals include mammals, and particularly humans. Other preferredanimals include murines, bovines, equines, porcines, canines, andfelines. The composition may include partially or significantly purifiedMSCRAMM-derived peptide epitopes, obtained from natural or recombinantsources, which proteins or peptides may be obtainable naturally oreither chemically synthesized, or alternatively produced in vitro fromrecombinant host cells expressing DNA segments encoding such epitopes.Smaller peptides that include reactive epitopes, such as those betweenabout 30 and about 100 amino acids in length will often be preferred.The antigenic proteins or peptides may also be combined with otheragents, such as other staphylococcal or streptococcal peptide or nucleicacid compositions, if desired. The composition may also includestaphylococcal produced bacterial components such as those discussedabove, obtained from natural or recombinant sources, which proteins maybe obtainable naturally or either chemically synthesized, oralternatively produced in vitro from recombinant host cells expressingDNA segments encoding such peptides.

Immunoformulations of this invention, whether intended for vaccination,treatment, or for the generation of antibodies useful in the detectionof staphylococci and streptococci, or prevention of bacterial adhesionto ECM components such as fibronectin, collagen, elastin, fibrinogen orvitronectin may comprise site-specifically mutated, truncated, orsynthetically-derived antigenic peptide fragments from these proteins.As such, antigenic functional equivalents of the proteins and peptidesdescribed herein also fall within the scope of the present invention.

Further means contemplated by the inventors for generating an immuneresponse in an animal includes administering to the animal, or humansubject, a pharmaceutically-acceptable composition comprising animmunologically effective amount of a nucleic acid composition encodinga peptide epitope, or an immunologically effective amount of anattenuated live organism that includes and expresses such a nucleic acidcomposition.

The amount of expressible DNA or transcribed RNA to be introduced into avaccine recipient will have a very broad dosage range and may depend onthe strength of the transcriptional and translational promoters used. Inaddition, the magnitude of the immune response may depend on the levelof protein expression and on the immunogenicity of the expressed geneproduct. In general, effective dose ranges of about 1 ng to 5 mg, 100 ngto 2.5 mg, 1 μg to 750 μg, and preferably about 10 μg to 300 μg of DNAis administered directly into muscle tissue. Subcutaneous injection,intradermal introduction, impression through the skin, and other modesof administration such as intraperitoneal, intravenous, or inhalationdelivery are also suitable. It is also contemplated that boostervaccinations may be provided. Following vaccination with an MSCRAMMpolynucleotide immunogen, boosting with MSCRAMM protein immunogens suchas the M55 gene product is also contemplated.

The polynucleotide may be “naked”, that is, unassociated with anyproteins, adjuvants or other agents which affect the recipients' immunesystem. In this case, it is desirable for the polynucleotide to be in aphysiologically acceptable solution, such as, but not limited to,sterile saline or sterile buffered saline. Alternatively, the DNA may beassociated with liposomes, such as lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture, or the DNA may beassociated with an adjuvant known in the art to boost immune responses,such as a protein or other carrier. Agents which assist in the cellularuptake of DNA, such as, but not limited to, calcium ions, may also beused. These agents are generally referred to herein as transfectionfacilitating reagents and pharmaceutically acceptable carriers.Techniques for coating microprojectiles coated with polynucleotide areknown in the art and are also useful in connection with this invention.For DNA intended for human use it may be useful to have the final DNAproduct in a pharmaceutically acceptable carrier or buffer solution.Pharmaceutically acceptable carriers or buffer solutions are known inthe art and include those described in a variety of texts such asRemington's Pharmaceutical Sciences.

In another embodiment, the invention is a polynucleotide which comprisescontiguous nucleic acid sequences capable of being expressed to producea gene product upon introduction of said polynucleotide into eukaryotictissues in vivo. The encoded gene product preferably either acts as animmunostimulant or as an antigen capable of generating an immuneresponse. Thus, the nucleic acid sequences in this embodiment encode anMSCRAMM immunogenic epitope, and optionally a cytokine or a T-cellcostimulatory element, such as a member of the B7 family of proteins.

There are several advantages of immunization with a gene rather than itsgene product. The first is the relative simplicity with which native ornearly native antigen can be presented to the immune system. Mammalianproteins expressed recombinantly in bacteria, yeast, or even mammaliancells often require extensive treatment to insure appropriateantigenicity. A second advantage of DNA immunization is the potentialfor the immunogen to enter the MHC class I pathway and evoke a cytotoxicT cell response. Immunization of mice with DNA encoding the influenza Anucleoprotein (NP) elicited a CD8⁺ response to NP that protected miceagainst challenge with heterologous strains of flu. (Montgomery, D. L.et al., Cell Mol Biol, 43(3):285-292, 1997; Ulmer, J. et al., Vaccine,15(8):792-794, 1997)

Cell-mediated immunity is important in controlling infection. Since DNAimmunization can evoke both humoral and cell-mediated immune responses,its greatest advantage may be that it provides a relatively simplemethod to survey a large number of S. aureus genes for their vaccinepotential.

Immunization by DNA injection also allows the ready assembly ofmulticomponent subunit vaccines. Simultaneous immunization with multipleinfluenza genes has recently been reported. (Donnelly, J. et al.,Vaccines, 55-59, 1994). The inclusion in a S. aureus vaccine of geneswhose products activate varied and mulitple responses from the immunesystem may also provide thorough protection from subsequent challenge.

Further provided is a composition comprising an isolated nucleic acidsegment that encodes a peptide of a binding domain of a binding protein,wherein the peptide does or does not specifically bind to its ligand. Itis also contemplated that attenuated organisms may be engineered toexpress recombinant MSCRAMM gene products and themselves be deliveryvehicles for the invention. Particularly preferred are attenuatedbacterial species such as Mycobacterium, and in particular M bovis, Msmegmatis, or BCG. Alternatively, pox-, polio-, adeno-, or otherviruses, and bacteria such as Salmonella, Shigella, Listeria,Streptococcus species may also be used in conjunction with the methodsand compositions disclosed herein.

The naked DNA technology, often referred to as genetic immunization, hasbeen shown to be suitable for protection against infectious organisms.Such DNA segments could be used in a variety of forms including nakedDNA and plasmid DNA, and may administered to the subject in a variety ofways including parenteral, mucosal, and so-called microprojectile-based“gene-gun” inoculations. The use of nucleic acid compositions of thepresent invention in such immunization techniques is thus proposed to beuseful as a vaccination strategy against at least streptococcal andstaphylococcal infection.

It is recognized by those skilled in the art that an optimal dosingschedule of a DNA vaccination regimen may include as many as five tosix, but preferably three to five, or even more preferably one to threeadministrations of the immunizing entity given at intervals of as few astwo to four weeks, to as long as five to ten years, or occasionally ateven longer intervals.

Particular aspects of the invention concern the use of plasmid vectorsfor the cloning and expression of recombinant peptides, and particularpeptide epitopes comprising either native, or site-specifically mutatedbinding site epitopes. The generation of recombinant vectors,transformation of host cells, and expression of recombinant proteins iswell-known to those of skill in the art. Prokaryotic hosts are preferredfor expression of the peptide compositions of the present invention. Anexample of a preferred prokaryotic host is E. coli, and in particular,E. coli, strains ATCC 69791, BL21(DE3), JMIOI, XL1-Blue™, RRI, LE392, B,^(χ776) (ATCC 31537), and W3110 (F, λ, prototrophic, ATCC 273325).Alternatively, other Enterobacteriaceae species such as Salmonellatyphimurium and Serratia marcescens, or even other Gram-negative hostsincluding various Pseudomonas species may be used in the recombinantexpression of the genetic constructs discussed herein. Additional hostsmay include well known eukaryotic and prokaryotic hosts, such as strainsof Bacillus, Streptomyces, fungi such as yeasts, and animal cells, suchas CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells(e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9),and human cells and plant cells in tissue culture.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA. Useful expression vectors, for example, may consistof segments of chromosomal, non-chromosomal and synthetic DNA sequences.Suitable vectors include derivatives of SV40 and known bacterialplasmids. e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and theirderivatives, plasmids such as RP4; phage DNAs, e.g., the numerousderivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2μplasmid or derivatives thereof; vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

As is well known in the art, DNA sequences may be expressed byoperatively linking them to an expression control sequence in anappropriate expression vector and employing that expression vector totransform an appropriate unicellular host. Such operative linking of aDNA sequence of this invention to an expression control sequence, ofcourse, includes, if not already part of the DNA sequence, the provisionof an initiation codon, ATG, in the correct reading frame upstream ofthe DNA sequence.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phospho-glycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast a-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly withregard to potential secondary structures. Suitable unicellular hostswill be selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences encoding the componentsof this invention on fermentation or in large scale animal culture.

In certain embodiments, it is also contemplated that the nucleic acidsegments discussed herein will be used to transect appropriate hostcells. Technology for introduction of DNA into cells is well-known tothose of skill in the art. Four general methods for delivering a nucleicsegment into cells have been described: (1) chemical methods (Graham andVanDerEb, Virology, 54 (2):536-539, 1973); physical methods such asmicroinjection (Capecchi, Cell, 22(2):479-488, 1980), electroporation(Wong and Neuman, Biochim Biophys Res Commun, 107(2):584-587, 1982;Fromm et al., Proc Natl Acad Sci USA, 82(17):5824-5828, 1985) and thegene gun (Yang et al., Proc Natl Acad Sci USA, 87:4144-4148, 1990); (3)viral vectors (Eglitis and Anderson, Bio/techniques, 6(7):608-614,1988); and (4) receptor-mediated mechanisms (Wagner, et al., Proc NatlAcad Sci USA, 89(13):6099-6103, 1992).

DNA sequences encoding MSCRAMM can be prepared synthetically or cloned.The DNA sequence can be designed with the appropriate codons for theMSCRAMM amino acid sequence. In general, one will select preferredcodons for the intended host if the sequence will be used forexpression. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature, 292:756 (1981);Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem.,259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express MSCRAMM analogs. Alternatively, DNA encoding analogs can bemade by site-directed mutagenesis of native MSCRAMM genes or cDNAs, andanalogs can be made directly using conventional polypeptide synthesis. Ageneral method for site-specific incorporation of unnatural amino acidsinto proteins is described in Noren et al., Science, 244:182-188 (April1989). This method may be used to create analogs with unnatural aminoacids.

XII. Antisense Oligonucleotides and Ribozymes

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of the MSCRAMM at the translational level. This approachutilizes antisense nucleic acid and ribozymes to block translation of aspecific mRNA, either by masking the mRNA with an antisense nucleic acidor cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. In the cell, theyhybridize to that specific mRNA, forming a double stranded molecule. Thecell does not translate an mRNA in this double-stranded form. Therefore,antisense nucleic acids interfere with the expression of mRNA intoprotein. Oligomers of about fifteen nucleotides and molecules thathybridize to the AUG initiation codon will be particularly efficient,since they are easy to synthesize and are likely to pose fewer problemsthan larger molecules when introducing them into MSCRAMM-producingcells. Antisense methods have been used to inhibit the expression ofmany genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, Nature,334(6183):585-591, 1988) Tetrahymena-type ribozymes recognize four-basesequences, while “hammerhead”-type recognizes eleven- to eighteen-basesequences. The longer the recognition sequence, the more likely it is tooccur exclusively in the target mRNA species. Therefore, hammerhead-typeribozymes are preferable to Tetrahymena-type ribozymes for inactivatinga specific mRNA species, and eighteen base recognition sequences arepreferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for MSCRAMM and theirligands.

XIII. Pharmaceutical Compositions

A pharmaceutical composition is provided that comprises the bindingproteins, the peptides, the antibodies, or the nucleic acids asdescribed above optionally in combination with bacterial components, ina pharmaceutically acceptable excipient, in an effective amount to treatS. aureus infection. The compositions are typically used in thepreparation of an immunization formulation that optionally includes anadjuvant and other customary additives. The compositions can alsocomprise diagnostic kits as described herein.

Methods for preparing pharmaceutical compositions which containpolypeptides, analogs or active fragments as active ingredients are wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions, however, solidforms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.The active therapeutic ingredient is often mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof Inaddition if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents which enhance the effectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of MSCRAMM binding capacity desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations often nanomolar to ten micromolar in the blood arecontemplated.

The therapeutic compositions may further include an effective amount ofthe MSCRAMM/MSCRAMM antagonist or analog thereof, and one or more of thefollowing active ingredients: an antibiotic, a steroid.

The preparation of vaccines that contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables, either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients that are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjutants that enhance the effectiveness of the vaccines.

The preparation of such compositions that are essentially free fromendotoxin can be achieved by following the published methodology, forexample, U.S. Pat. No. 4,271,147 (incorporated herein by reference)discloses methods for the preparation of Neisseria meningitides membraneproteins for use in vaccines.

The immunological compositions, such as vaccines, and otherpharmaceutical compositions can be used alone or in combination withother blocking agents to protect against human and animal infectionscaused by staphylococcal bacteria such as S. aureus. In particular, thecompositions can be used to protect humans against endocarditis or toprotect humans or ruminants against mastitis caused by staphylococcalinfections. The vaccine can also be used to protect canine and equineanimals against similar staphylococcal infections.

To enhance immunogenicity, the proteins may be conjugated to a carriermolecule. Suitable immunogenic carriers include proteins, polypeptidesor peptides such as albumin, hemocyanin, thyroglobulin and derivativesthereof, particularly bovine serum albumin (BSA) and keyhole limpethemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solidphases. Other protein derived or non-protein derived substances areknown to those skilled in the art. An immunogenic carrier typically hasa molecular weight of at least 1,000 daltons, preferably greater than10,000 daltons. Carrier molecules often contain a reactive group tofacilitate covalent conjugation to the hapten. The carboxylic acid groupor amine group of amino acids or the sugar groups of glycoproteins areoften used in this manner. Carriers lacking such groups can often bereacted with an appropriate chemical to produce them. Preferably, animmune response is produced when the immunogen is injected into animalssuch as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, andother animals, most preferably mice and rabbits. Alternatively, amultiple antigenic peptide comprising multiple copies of the protein orpolypeptide, or an antigenically or immunologically equivalentpolypeptide may be sufficiently antigenic to improve immunogenicitywithout the use of a carrier.

The MSCRAMM protein or proteins may be administered with an adjuvant inan amount effective to enhance the immunogenic response against theconjugate. At this time, the only adjuvant widely used in humans hasbeen alum (aluminum phosphate or aluminum hydroxide). Saponin and itspurified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines. However, chemicallydefined preparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal. (J. Immunol. 147:410-415, 1991) and incorporated by referenceherein, encapsulation of the conjugate within a proteoliposome asdescribed by Miller et al., (J. Exp. Med. 176:1739-1744, 1992) andincorporated by reference herein, and encapsulation of the protein inlipid vesicles such as Novasome™ lipid vesicles (Micro Vescular Systems,Inc., Nashua, N.H.) may also be useful.

In certain embodiments, the inventors contemplate the use of liposomesand/or nanocapsules for the introduction of particular peptides ornucleic acid segments into host cells. In particular, the malonyltyrosyland phosphotyrosyl peptides of the present invention may be formulatedfor delivery in solution with DMSO or encapsulated in liposomes.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids, peptides,and/or antibodies disclosed herein. The formation and use of liposomesis generally known to those of skill in the art (see for example,Couvreur et al, FEBS Lett, 84:323-326, 1977; and Crit Rev Ther DrugCarrier Syst, 5:1-20, 1988 which describes the use of liposomes andnanocapsules in the targeted antibiotic therapy of intracellularbacterial infections and diseases). Recently, liposomes were developedwith improved serum stability and circulation half-times (Gabizon andPapahadjopoulos, Proc Natl Acad Sci USA, 85:6949-6953, 1988; Allen andChoun, FEBS Lett, 223:42-46, 1987).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Muller etal., DNA Cell Biol, 9(3):221-229, 1990). In addition, liposomes are freeof the DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs,radiotherapeutic agents, enzymes, viruses, transcription factors andallosteric effectors into a variety of cultured cell lines and animals.In addition, several successful clinical trails examining theeffectiveness of liposome-mediated drug delivery have been completed(Lopez-Berestein et al, Cancer Drug Review, 2(3):183-189, 1985; Sculieret al, Eur J Cancer Clin Oncol, 24(3):527-538, 1988). Furthermore,several studies suggest that the use of liposomes is not associated withautoimmune responses, toxicity or gonadal localization after systemicdelivery (Mori and Fukatsu, Epilepsia, 33(6):994-1000, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 micron. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVS) with diameters in therange of 200 to 500 A, containing an aqueous solution in the core.

Liposomes bear many resemblances to cellular membranes and arecontemplated for use in connection with the present invention ascarriers for the peptide compositions. They are widely suitable, as bothwater- and lipid-soluble substances can be entrapped, i.e., in theaqueous spaces and within the bilayer itself, respectively. It ispossible that the drug-bearing liposomes may even be employed forsite-specific delivery of active agents by selectively modifying theliposomal formulation.

In addition to the teachings of Couvreur et al. (FEBS Lett,84:323-326,1977; and Crit Rev Ther Drug Carrier Syst, 5:1-20, 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins such as cytochrome Cbind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVS.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depends ontheir physical properties, such as size, fluidity and surface charge.They may persist in tissues for hours or days, depending on theircomposition, and half lives in the blood range from minutes to severalhours. Larger liposomes, such as MLVs and LUVS, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the peptides of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al, Int J. Pharm, 35:121-127, 1987). To avoidside effects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 micron) should be designed using polymersable to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention, and such particles may be easily made, asdescribed by Couvreur et al, (supra, 1977 and1988).

Suitable methods of administration include, but are not limited to,topical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal and intradermal administration.

For topical administration, the composition is formulated in the form ofan ointment, cream, gel, lotion, drops (such as eye drops and eardrops), or solution (such as mouthwash). Wound or surgical dressings,sutures and aerosols may be impregnated with the composition. Thecomposition may contain conventional additives, such as preservatives,solvents to promote penetration, and emollients. Topical formulationsmay also contain conventional carriers such as cream or ointment bases,ethanol, or oleyl alcohol.

In a preferred embodiment, a vaccine is packaged in a single dosage forimmunization by parenteral (i.e., intramuscular, intradermal orsubcutaneous) administration or nasopharyngeal (i.e., intranasal)administration. The vaccine is most preferably injected intramuscularlyinto the deltoid muscle. The vaccine is preferably combined with apharmaceutically acceptable carrier to facilitate administration. Thecarrier is usually water or a buffered saline, with or without apreservative. The vaccine may be lyophilized for resuspension at thetime of administration or in solution.

The carrier to which the protein may be conjugated may also be apolymeric delayed release system. Synthetic polymers are particularlyuseful in the formulation of a vaccine to effect the controlled releaseof antigens. For example, the polymerization of methyl methacrylate intospheres having diameters less than one micron has been reported byKreuter, J., Microcapsules And Nanoparticles In Medicine AndPharmacology, M. Donbrow (Ed). CRC Press, p. 125-148.

Microencapsulation of the protein will also give a controlled release. Anumber of factors contribute to the selection of a particular polymerfor microencapsulation. The reproducibility of polymer synthesis and themicroencapsulation process, the cost of the microencapsulation materialsand process, the toxicological profile, the requirements for variablerelease kinetics and the physicochemical compatibility of the polymerand the antigens are all factors that must be considered. Examples ofuseful polymers are polycarbonates, polyesters, polyurethanes,polyorthoesters polyamides, poly (d,l-lactide-co-glycolide) (PLGA) andother biodegradable polymers. The use of PLGA for the controlled releaseof antigen is reviewed by Eldridge, J. H., et al. Current Topics InMicrobiology And Immunology, 146:59-66 (1989).

The preferred dose for human administration is from 0.01 mg/kg to 10mg/kg, preferably approximately 1 mg/kg. Based on this range, equivalentdosages for heavier body weights can be determined. The dose should beadjusted to suit the individual to whom the composition is administeredand will vary with age, weight and metabolism of the individual. Thevaccine may additionally contain stabilizers such as thimerosal(ethyl(2-mercaptobenzoate-S) mercury sodium salt) (Sigma ChemicalCompany, St. Louis, Mo.) or physiologically acceptable preservatives.

It will be readily apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

XIV. Kits

This invention also includes a kit comprising anti-MSCRAMM antibody oran MSCRAMM antigen for the detection and diagnosis of infections causedor exacerbated by Staphylococcus bacteria such as S. aureus or S.epidermidis. The preferred kit contains sufficient antibody to bindsubstantially all of the antigen in the sample in about ten minutes orless, or sufficient antigen to bind antibodies for MSCRAMMs. Theantibody or antigen can be immobilized on a solid support, and can belabeled with a detectable agent, as described above. The kit optionallycontains a means for detecting the detectable agent. If the antibody orantigen is labeled with a fluorochrome or radioactive label, no meansfor detecting the agent will typically be provided, as the user will beexpected to have the appropriate spectrophotometer, scintillationcounter, or microscope. If the detectable agent is an enzyme, a meansfor detecting the detectable agent can be supplied with the kit, andwould typically include a substrate for the enzyme in sufficientquantity to detect all of the antigen-antibody complex. One preferredmeans for detecting a detectable agent is a substrate that is convertedby an enzyme into a colored product. A common example is the use of theenzyme horseradish peroxidase with2,2′-azino-di-[3-ethyl-benzothiazoline sulfonate] (ABTS).

The kit can optionally contain a lysing agent that lyses cells presentin the sample of body fluid. Suitable lysing agents include surfactantssuch as Tween-80, Nonidet P40, and Triton X-100. Preferably, the lysingagent is immobilized onto the solid support along with the antibody.

The kit can also contain a buffer solution for washing the substratebetween steps. The buffer solution is typically a physiological solutionsuch as a phosphate buffer, physiological saline, citrate buffer, orTris buffer.

The kit can optionally include different concentrations of a preformedantigen to calibrate the assay. The kit can additionally contain avisual or numeric representation of amounts of antigen in a calibratedstandard assay for reference purposes. For example, if an assay is usedthat produces a colored product, a sheet can be included that provides adepiction of increasing intensities associated with differing amounts ofantigen.

The kit can optionally include two antibodies in the detection system.The first antibody which is present in small amounts is specific for theantigen being assayed for. The second antibody provided in higheramounts is used to detect the first antibody. For example, a rabbitantibody can be used to detect the LOOH/amine antigen, and then ananti-rabbit IgG antibody can be used to detect the bound rabbitantibody. Goat antibodies and anti-antibodies are also commonly used.

As one nonlimiting example, a kit for the detection of the lipidperoxidation state of a patient is provided that includes a rabbitantibody specific for desired antibody, anti-rabbit IgG antibody insufficient amounts to detect the bound first antibody, an enzymeconjugated to the second antibody and a substrate for the enzyme whichchanges color on exposure to the enzyme. In addition, a kit may beprepared using one or more MSCRAMM antigens such as the M55 domain ofthe collagen binding protein and the ClfA fibrinogen binding protein,and this kit will enable the detection of samples with antibodies tocollagen binding and fibrinogen binding MSCRAMMs.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing form the spirit andscope of the invention.

Example 1 Preparation of Prototype Four Component MSCRAMM Vaccine

A series of recombinant proteins, representing domains from thecollagen, Fn, and Fbg-binding MSCRAMMs (FIG. 1), were overexpressed inE. coli and affinity purified by metal chelating chromatography aspreviously described (see, e.g., Joh et al., Biochemistry. 33(20):6086-6092, 1994; Patti et al., J. Biol. Chem. 270, 12005-12011,1995; McDevitt et al., Mol. Micro. 11 (2):237-248, 1994; Ni Eidhin etal., Infect. Immun. Submitted, 1998). Used were the following: aminoacids contained in the recombinant collagen-binding MSCRAMM expressedfrom cna (M55, such as disclosed in co-pending U.S. patent applicationSer. No. 08/856,253, incorporated herein by reference); amino acidscontained in the recombinant fibrinogen-binding MSCRAMM expressed fromclfA (pCF40, such as disclosed in U.S. patent application Ser. No.08/293,728, incorporated herein by reference); amino acids contained inthe recombinant fibrinogen-binding MSCRAMM expressed from clfB (RegionA, such as disclosed in U.S. application Ser. No. 09/200,650,incorporated herein by reference); and amino acids contained in therecombinant fibronectin-binding MSCRAMM (DUD4, such as those disclosedin co-pending U.S. application Ser. No. 09/010,317, incorporated hereinby reference). The recombinant FN-binding MSCRAMM protein DUD4 wastreated with formalin (5% formalin overnight, 4° C.) prior to combiningit with the M55, Region A from ClfA, and Region A from ClfB.

Example 2 Example of growing E. coli Strains for Production ofRecombinant Proteins

Overnight cultures of E. coli JM101 or TOP 3 cells (Stratagene)harboring the recombinant plasmids were diluted 1:50 in 1 L of LuriaBroth (Gibco BRL) containing 50 mg/mL ampicillin. E. coli cells weregrown until the culture reached an OD₆₀₀ of 0.5-0.8. Expression of therecombinant proteins was induced by adding IPTG to a final concentrationof 0.2 mM. After a three hour induction period, cells were collected bycentrifugation, resuspended in 15 mL of Buffer A (5 mM imidazole, 0.5 MNaCl, 20 mM Tris-HCl, pH 7.9) and lysed by passage through a Frenchpress twice at 20,000 lb./in². Cell debris was removed by centrifugationat 50,000×g for 10 min and the supernatant was passed through a 0.45 μMfilter.

Example 3 Purification of HIS₆ Containing Recombinant Proteins Expressedfrom pQE-30 (Qiagen®; Qiagen Inc. Chatsworth. Calif.) or PV-4 BasedRecombinant Plasmids

The recombinant proteins were purified by immobilized metal chelatechromatography, using a column of iminodiacetic acid/Sepharoses® 6B FastFlow (Sigma, St. Louis, Mo.) charged with Ni²⁺; (Porath et al. 1975;Hochuli et al. 1988). The HIS₆ tagged proteins were purified byimmobilized metal chelate affinity chromatography. More specifically, acolumn containing iminodiacetic acid Sepharoset® 6B FF, connected to aFPLC® system (Pharmacia), was charged with 150 mM Ni⁺⁺ and equilibratedwith buffer A (5 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). Afterequilibration, the bacterial supernatant was applied to the column andthe column was washed with 10 bed volumes of buffer A. Subsequently, thecolumn was eluted with buffer B (200 mM imidazole, 0.5 M NaCl, 20 mMTris, pH 7.9). The eluate was monitored for protein by the absorbance at280 nm and peak fractions were analyzed by SDS-PAGE. Endotoxin wasremoved from the purified recombinant proteins by detergent extractionwith 1% Triton X-114 followed by metal chelate affinity chromatographyand passage through a polymyxcin B-sepharose column. The level ofendotoxin was quantitated using a chromogenic Limulus Amebocyte Lysate(BioWhittaker, Walkersville, Md.) assay.

Example 4 Immunization of Animals with Four Component MSCRAMMVaccine—MSCRAMM IV Rhesus Monkeys

100 μg of M55 (1 EU/mg), ClfA (2.5 EU/mg), ClfB (<1.0 EU/mg), and DUD4(<10 EU/mg) were mixed together to form the MSCRAMM IV vaccine. Thecocktail was mixed with TiterMax™ Gold (CytRX, Norcross, Ga.) in a 1:1ratio. Two female rhesus monkeys, ID#495Z & 664U (˜9.4 kg), werevaccinated intramuscularly (IM) in the hind quadricep with 200 μl of thevaccine. Twenty-eight days later the two monkeys were boosted IM with200 μl of the same vaccine formulation. Two additional female monkeys,ID#215W & 203U (˜8.0 kg), were immunized with the MSCRAMM IV that wascompounded in a 1:1 ratio with aluminum hydroxide (2% Alhydrogel;Superfos, Denmark). Twenty-eight days later the two monkeys were boostedIM with 200 μl of the same vaccine formulation. The clinical regimenfollowed is described below.

Day 0 15 ml pre-immunization plasma sample, complete blood chemistry Day1 Vaccinate IM hind quadricep with 0.2 ml MSCRAMM IV (100 μg), injectionsite exam, temperature recorded Day 7 Liver panel, temperature recorded,injection site exam Day 14 15 ml plasma sample Day 21 15 ml plasmasample Day 28 Complete blood chemistry, temperature recorded 15 mlplasma sample, boost with IM injection of 0.2 ml MSCRAMM IV (100 μg) Day30 Liver panel, temperature recorded, injection site exam Day 35 Liverpanel, temperature recorded, injection site, 15 ml plasma sample Day 4215 ml plasma sample Day 49 15 ml plasma sample Day 106 15 ml plasmasample

All 4 animals seroconverted following the initial immunization. Antibodylevels >3 times above background could be detected by ELISA 106 daysafter the primary vaccination. The four animals received another boosterimmunization in the 21^(st) week of the study. Each animal was given abooster of four subcutaneous injections of 125 μl of the vaccine for atotal booster of 600 μl of the vaccine. Antibody levels at least 3 timesabove background, and as much as 15 times above background, could bedetected by ELISA 189 days after the primary vaccination. See FIG. 2. Noadverse injection site reactions were detected by direct observation byveterinarians. In addition, liver enzyme profiles, CBC, and hematologyprofiles were within the normal range for rhesus monkeys.

Example 5 Analysis of Plasma Samples from the Vaccinated Monkeys wereAnalyzed by ELISA

Immulon-2 microtiter plates (Dynex Technologies, Chantilly, Va.) werecoated overnight at 4° C. with 10 μg/ml (50 μl) of the collagen bindingMSCRAMM (M55), fibrinogen binding MSCRAMM (clfA; pCF44), fibrinogenbinding MSCRAMM (ClfB; Region A), and the fibronectin binding MSCRAMM(DUD4). Fifty microliters of the diluted plasma samples were added tothe MSCRAMM coated wells and incubated for 1 hr at room temperature.Wash buffer consisting of PBS containing 0.05% vol/vol Tween-20, ablocking solution of 1% wt/vol BSA, 0.05% Tween-20 in PBS, and antibodydilution buffer consisting of PBS containing 0.1% BSA, 0.05% Tween-20.Incubation with primary and secondary antibodies was for 60 min at 25°C. The secondary antibody was alkaline phosphatase-conjugated goatanti-monkey immunoglobulin G, (Rockland, Gilbertsville, Pa.), diluted3500-fold in antibody dilution buffer. ELISA plates were developed for30 min at 37° C. with 1 mg/ml p-nitrophenyl phosphate (Sigma) in 1 Mdiethanolamine, 0.5 mM MgCl₂, pH 9.8, and quantified at 405 nm on aPerkin Elmer HTS 7000 Bio-Assay reader. Each plasma sample was diluted100-fold in phosphate buffered saline, containing 0.05% Tween 20, 0.1%BSA, pH 7.4. ELISA data are shown in FIG. 2.

Example 6 Inhibition Assays

Methicillin resistant S. aureus strain 601 (Smeltzer, M. S., Gene.196:249-159, 1997) was cultured under constant rotation for 15 h at 37°C. in BHI broth. A 1:100 dilution of the overnight culture was made intoBHI and the bacteria were grown at 37° C. until mid exponential phase.The bacteria were harvested by centrifugation, washed three times insterile PBS, pH 7.4, and then resuspended in a carbonate buffer (50 mMNaHCO₃, pH 8.5). The bacteria were mixed with 1 mg/ml FITC (Sigma;F-7250) in 50 mM NaHCO₃, pH 8.5 and incubated end-over-end in the darkfor 1 hr at 25° C. The FITC labeling reaction was stopped bycentrifugation of the bacterial cells and removing the supernatantcontaining the unreacted FITC. The labeled bacteria were washed threetimes in PBS to remove unincorporated FITC, resuspended in PBS, adjustedto ˜1×10⁸ cfu/ml and stored at −20° C. in PBS, pH 7.4.

Example 7 Purification of IgG from Immunized Monkeys

IgG was purified from the monkey plasma by affinity chromatography onPROSEP®-A high capacity resin (Bioprocessing Inc., Princeton, N.J.).Briefly, the plasma was thawed and passed through 0.45μ filter. Theplasma was applied to a benchtop column containing PROSEPT®-A highcapacity resin. The unbound material was removed by washing the columnextensively with PBS. The IgG was eluted from the column with 0.1 Msodium citrate, pH 3.0. The pH of eluted IgG was immediately neutralizedto pH 6.8-7.4 by the addition of 1M Tris, pH 9.0. The IgG was thendialyzed into PBS, pH 7.4, concentrated and filter sterilized. Theconcentration of the purified IgG was determined by absorbance at 280nm.

Example 8 Competitive Inhibition ELISA

Costar 96 well black plates were coated overnight at 4° C. or at roomtemperature for 2 hr with a 10 μg/ml solution of matrix componentsconsisting of bovine collagen, human fibrinogen, and bovine fibronectinin PBS, pH 7.4. The matrix protein coated plates were washed three timeswith PBS, 0.05% Tween 20 and then blocked with PBS, 1% BSA. The blockedplates were washed three times with PBS, 0.05% Tween 20. A 500 μlaliquot of FITC-labeled S. aureus cells were mixed with an increasingamount of purified monkey IgG in PBS, 0.05% Tween 20, 0.1%BSA. Thelabeled cells and IgG were mixed on an end-over-end shaker for 1 hr at25° C. Fifty μl of the labeled cells/IgG mixture was added to each wellon the microtiter plate and incubated at 25° C. on a rocker platform.The wells were washed three times with PBS, 0.05% Tween 20. The amountof bacteria bound to the immobilized matrix proteins was determined on aPerkin Elmer HTS 7000 Bio-Assay reader with the excitation filter set at485 nm and the emission filter set at 535 nm.

Example 2 Animal Model of Sepsis

Using a mouse model of sepsis (Bremell, T. A., et al., Infect. Immun. 62(7):2976-2985, 1992) we have demonstrated that passive immunization withIgG purified from rhesus monkeys immunized with the MSCRAMM IV canprotect mice against sepsis induced death. Naive male NMRI mice 5-8weeks old were passively immunized i.p. on day-1 with 20 mg of eitherpurified IgG from rhesus monkeys immunized with MSCRAMM IV (n=12), orIgG from non-immunized rhesus monkeys (n=13). On day 0, the mice werechallenged i.v. with 2.4×10⁷ CFU/mouse S. aureus strain LS-1. Mortalityand weight change was monitored over the next 3 days. Three days afterthe inoculation 3/13 mice (13%) were dead in the control group, comparedto 0/12 mice (0%) in the control group. Mortality in control group atday 13 was 53.8% (7/13) compared to only 16.2% (2/12) for the MSCRAMM IVpassively immunized group. The control mice exhibited a significantdecrease in their body weight compared to MSCRAMM IV IgG passivelyimmunized mice (28.0±2.5% vs 21.3±3.1%; p<0.01).

Example 10 Multicomponent Vaccines Containing M55 (Collagen-BindingMSCRA and ClfA (Fibrinogen-binding MSCRAMM)

Sixty female Swiss Webster mice received a total of 50 μg of eitherovalbumin, M55 (collagen-binding MSCRAMM) or a combination of M55 andClfA (fibrinogen-binding MSCRAM) proteins via a subcutaneous injection.The primary injection was prepared by emulsifying the antigens inFreund's Complete Adjuvant. The mice received a second injection of 25μg total protein in Freund's Incomplete Adjuvant 14 days after theprimary injection. A final injection of 25 μg total protein in PBS wasgiven 28 days after the primary injection. Post bleed samples from allmice were obtained two weeks after the final injection to determineantibody titers against the different MSCRAMM proteins. The mice were-then challenged (42 days after primary injection) via a singleintravenous injection with 1.2×10⁸ CFU of S. aureus 601. At day 5post-challenge, the mice were sacrificed and their kidneys harvested.The kidneys were then homogenized and plated on blood agar plates. Theplates were incubated at 37° C. overnight and the bacterial load in thekidneys was determined by colony counts. The results of the experimentshowed a two log difference in bacterial load between the ovalbumingroup (7.03±0.93 log CFU/g) and the M55/ClfA group (4.83±3.04 log CFU/g,p=0.006). A difference in bacterial load was also observed in the M55group (5.86±3.42 log CFU/g, p=0.003) when compared to the ovalbumingroup.

As shown in the above specification and examples, immunologicalcompositions, including vaccines, and other pharmaceutical compositionscontaining the MSCRAMM proteins are included within the scope of thepresent invention. One or more of the binding proteins, or active orantigenic fragments thereof, or fusion proteins thereof can beformulated and packaged, alone or in combination with other antigens,using methods and materials known to those skilled in the art forvaccines. The immunological response may be used therapeutically orprophylactically and may provide antibody immunity or cellular immunitysuch as that produced by T lymphocytes such as cytotoxic T lymphocytesor CD4+ T lymphocytes.

18 1 5406 DNA Staphylococcus epidermidis CDS (1)..(5406) 1 tat tgg ataaat tat gct tat aaa gta ttt aca taa aaa tgt aaa tgc 48 Tyr Trp Ile AsnTyr Ala Tyr Lys Val Phe Thr Lys Cys Lys Cys 1 5 10 15 aat tta caa gtaaat att caa att att tcc ttg taa aat att tat ttt 96 Asn Leu Gln Val AsnIle Gln Ile Ile Ser Leu Asn Ile Tyr Phe 20 25 30 aac tgg agg tat agt atgaaa aag aga aga caa gga cca att aac aag 144 Asn Trp Arg Tyr Ser Met LysLys Arg Arg Gln Gly Pro Ile Asn Lys 35 40 45 aga gtg gat ttt cta tcc aacaag gta aac aag tac tcg att agg aag 192 Arg Val Asp Phe Leu Ser Asn LysVal Asn Lys Tyr Ser Ile Arg Lys 50 55 60 ttc aca gta ggt aca gct tca atactc gtg ggt gct acg tta atg ttt 240 Phe Thr Val Gly Thr Ala Ser Ile LeuVal Gly Ala Thr Leu Met Phe 65 70 75 80 ggt gcc gca gac aat gag gct aaagcg gct gaa gac aat caa tta gaa 288 Gly Ala Ala Asp Asn Glu Ala Lys AlaAla Glu Asp Asn Gln Leu Glu 85 90 95 tca gct tca aaa gaa gaa cag aaa ggtagt cgt gat aat gaa aac tca 336 Ser Ala Ser Lys Glu Glu Gln Lys Gly SerArg Asp Asn Glu Asn Ser 100 105 110 aaa ctt aat caa gtc gat tta gac aacgga tca cat agt tct gag aaa 384 Lys Leu Asn Gln Val Asp Leu Asp Asn GlySer His Ser Ser Glu Lys 115 120 125 aca aca aat gta aac aat gca act gaagta aaa aaa gtt gaa gca cca 432 Thr Thr Asn Val Asn Asn Ala Thr Glu ValLys Lys Val Glu Ala Pro 130 135 140 acg aca agt gac gta tct aag cct aaagct aat gaa gca gta gtg acg 480 Thr Thr Ser Asp Val Ser Lys Pro Lys AlaAsn Glu Ala Val Val Thr 145 150 155 160 aat gag tca act aaa cca aaa acaaca gaa gca cca act gtt aat gag 528 Asn Glu Ser Thr Lys Pro Lys Thr ThrGlu Ala Pro Thr Val Asn Glu 165 170 175 gaa tca ata gct gaa aca ccc aaaacc tca act aca caa caa gat tcg 576 Glu Ser Ile Ala Glu Thr Pro Lys ThrSer Thr Thr Gln Gln Asp Ser 180 185 190 act gag aag aat aat cca tct ttaaaa gat aat tta aat tca tcc tca 624 Thr Glu Lys Asn Asn Pro Ser Leu LysAsp Asn Leu Asn Ser Ser Ser 195 200 205 acg aca tct aaa gaa agt aaa acagac gaa cat tct act aag caa gct 672 Thr Thr Ser Lys Glu Ser Lys Thr AspGlu His Ser Thr Lys Gln Ala 210 215 220 caa atg tct act aat aaa tca aattta gac aca aat gac tct cca act 720 Gln Met Ser Thr Asn Lys Ser Asn LeuAsp Thr Asn Asp Ser Pro Thr 225 230 235 240 caa agt gag aaa act tca tcacaa gca aat aac gac agt aca gat aat 768 Gln Ser Glu Lys Thr Ser Ser GlnAla Asn Asn Asp Ser Thr Asp Asn 245 250 255 cag tca gca cct tct aaa caatta gat tca aaa cca tca gaa caa aaa 816 Gln Ser Ala Pro Ser Lys Gln LeuAsp Ser Lys Pro Ser Glu Gln Lys 260 265 270 gta tat aaa aca aaa ttt aatgat gaa cct act caa gat gtt gaa cac 864 Val Tyr Lys Thr Lys Phe Asn AspGlu Pro Thr Gln Asp Val Glu His 275 280 285 acg aca act aaa tta aaa acacct tct gtt tca aca gat agt tca gtc 912 Thr Thr Thr Lys Leu Lys Thr ProSer Val Ser Thr Asp Ser Ser Val 290 295 300 aat gat aag caa gat tac acacga agt gct gta gct agt tta ggt gtt 960 Asn Asp Lys Gln Asp Tyr Thr ArgSer Ala Val Ala Ser Leu Gly Val 305 310 315 320 gat tct aat gaa aca gaagca att aca aat gca gtt aga gac aat tta 1008 Asp Ser Asn Glu Thr Glu AlaIle Thr Asn Ala Val Arg Asp Asn Leu 325 330 335 gat tta aaa gct gca tctaga gaa caa atc aat gaa gca atc att gct 1056 Asp Leu Lys Ala Ala Ser ArgGlu Gln Ile Asn Glu Ala Ile Ile Ala 340 345 350 gaa gca cta aaa aaa gacttt tct aac cct gat tat ggt gtc gat acg 1104 Glu Ala Leu Lys Lys Asp PheSer Asn Pro Asp Tyr Gly Val Asp Thr 355 360 365 cca tta gct cta aac agatct caa tca aaa aat tca cca cat aag agt 1152 Pro Leu Ala Leu Asn Arg SerGln Ser Lys Asn Ser Pro His Lys Ser 370 375 380 gca agt cca cgc atg aattta atg agt tta gct gct gag cct aat agt 1200 Ala Ser Pro Arg Met Asn LeuMet Ser Leu Ala Ala Glu Pro Asn Ser 385 390 395 400 ggt aaa aat gtg aatgat aaa gtt aaa atc aca aac cct acg ctt tca 1248 Gly Lys Asn Val Asn AspLys Val Lys Ile Thr Asn Pro Thr Leu Ser 405 410 415 ctt aat aag agt aataat cac gct aat aac gta ata tgg cca aca agt 1296 Leu Asn Lys Ser Asn AsnHis Ala Asn Asn Val Ile Trp Pro Thr Ser 420 425 430 aac gaa caa ttt aattta aaa gca aat tat gaa tta gat gac agc ata 1344 Asn Glu Gln Phe Asn LeuLys Ala Asn Tyr Glu Leu Asp Asp Ser Ile 435 440 445 aaa gag gga gat actttt act att aag tat ggt cag tat att aga ccg 1392 Lys Glu Gly Asp Thr PheThr Ile Lys Tyr Gly Gln Tyr Ile Arg Pro 450 455 460 ggt ggt tta gaa cttcct gca ata aaa act caa cta cgt agt aag gat 1440 Gly Gly Leu Glu Leu ProAla Ile Lys Thr Gln Leu Arg Ser Lys Asp 465 470 475 480 ggc tct att gtagct aat ggt gta tat gat aaa act aca aat acg acg 1488 Gly Ser Ile Val AlaAsn Gly Val Tyr Asp Lys Thr Thr Asn Thr Thr 485 490 495 act tat aca tttact aac tat gtt gat caa tat caa aat att aca ggt 1536 Thr Tyr Thr Phe ThrAsn Tyr Val Asp Gln Tyr Gln Asn Ile Thr Gly 500 505 510 agt ttt gat ttaatt gcg acg cct aag agg gaa aca gca att aag gat 1584 Ser Phe Asp Leu IleAla Thr Pro Lys Arg Glu Thr Ala Ile Lys Asp 515 520 525 aat cag aat tatcct atg gaa gtg acg att gct aac gaa gta gtc aaa 1632 Asn Gln Asn Tyr ProMet Glu Val Thr Ile Ala Asn Glu Val Val Lys 530 535 540 aaa gac ttc attgtg gat tat ggt aat aaa aag gac aat aca act aca 1680 Lys Asp Phe Ile ValAsp Tyr Gly Asn Lys Lys Asp Asn Thr Thr Thr 545 550 555 560 gca gcg gtagca aat gtg gat aat gta aat aat aaa cat aac gaa gtt 1728 Ala Ala Val AlaAsn Val Asp Asn Val Asn Asn Lys His Asn Glu Val 565 570 575 gtt tat ctaaac caa aat aac caa aac cct aaa tat gct aaa tat ttc 1776 Val Tyr Leu AsnGln Asn Asn Gln Asn Pro Lys Tyr Ala Lys Tyr Phe 580 585 590 tca aca gtaaaa aat ggt gaa ttt ata cca ggt gaa gtg aaa gtt tac 1824 Ser Thr Val LysAsn Gly Glu Phe Ile Pro Gly Glu Val Lys Val Tyr 595 600 605 gaa gtg acggat acc aat gcg atg gta gat agc ttc aat cct gat tta 1872 Glu Val Thr AspThr Asn Ala Met Val Asp Ser Phe Asn Pro Asp Leu 610 615 620 aat agt tctaat gta aaa gat gtg aca agt caa ttt gca cct aaa gta 1920 Asn Ser Ser AsnVal Lys Asp Val Thr Ser Gln Phe Ala Pro Lys Val 625 630 635 640 agt gcagat ggt act aga gtt gat atc aat ttt gct aga agt atg gca 1968 Ser Ala AspGly Thr Arg Val Asp Ile Asn Phe Ala Arg Ser Met Ala 645 650 655 aat ggtaaa aag tat att gta act caa gca gtg aga cca acg gga act 2016 Asn Gly LysLys Tyr Ile Val Thr Gln Ala Val Arg Pro Thr Gly Thr 660 665 670 gga aatgtt tat acc gaa tat tgg tta aca aga gat ggt act acc aat 2064 Gly Asn ValTyr Thr Glu Tyr Trp Leu Thr Arg Asp Gly Thr Thr Asn 675 680 685 aca aatgat ttt tac cgt gga acg aag tct aca acg gtg act tat ctc 2112 Thr Asn AspPhe Tyr Arg Gly Thr Lys Ser Thr Thr Val Thr Tyr Leu 690 695 700 aat ggttct tca aca gca cag ggg gat aat cct aca tat agt cta ggt 2160 Asn Gly SerSer Thr Ala Gln Gly Asp Asn Pro Thr Tyr Ser Leu Gly 705 710 715 720 gactat gta tgg tta gat aaa aat aaa aac ggt gtt caa gat gat gat 2208 Asp TyrVal Trp Leu Asp Lys Asn Lys Asn Gly Val Gln Asp Asp Asp 725 730 735 gagaaa ggt tta gca ggt gtt tat gtt act ctt aaa gac agt aac aat 2256 Glu LysGly Leu Ala Gly Val Tyr Val Thr Leu Lys Asp Ser Asn Asn 740 745 750 agagaa tta caa cgt gta act act gat caa tct gga cat tat caa ttt 2304 Arg GluLeu Gln Arg Val Thr Thr Asp Gln Ser Gly His Tyr Gln Phe 755 760 765 gataat tta caa aat gga acg tac aca gtc gag ttt gcg att cct gat 2352 Asp AsnLeu Gln Asn Gly Thr Tyr Thr Val Glu Phe Ala Ile Pro Asp 770 775 780 aattat acg cca tct ccc gca aat aat tct aca aat gat gca ata gat 2400 Asn TyrThr Pro Ser Pro Ala Asn Asn Ser Thr Asn Asp Ala Ile Asp 785 790 795 800tca gat ggt gaa cgt gat ggt aca cgt aaa gta gtt gtt gcc aaa gga 2448 SerAsp Gly Glu Arg Asp Gly Thr Arg Lys Val Val Val Ala Lys Gly 805 810 815aca att aat aat gct gat aat atg act gta gat act ggc ttt tat tta 2496 ThrIle Asn Asn Ala Asp Asn Met Thr Val Asp Thr Gly Phe Tyr Leu 820 825 830act cct aaa tac aat gtc gga gat tat gta tgg gaa gat aca aat aaa 2544 ThrPro Lys Tyr Asn Val Gly Asp Tyr Val Trp Glu Asp Thr Asn Lys 835 840 845gat ggt atc caa gat gac aat gaa aaa gga att tct ggt gtt aaa gta 2592 AspGly Ile Gln Asp Asp Asn Glu Lys Gly Ile Ser Gly Val Lys Val 850 855 860acg tta aaa aat aaa aat gga gat act att ggc aca acg aca aca gat 2640 ThrLeu Lys Asn Lys Asn Gly Asp Thr Ile Gly Thr Thr Thr Thr Asp 865 870 875880 tca aat ggt aaa tat gaa ttc aca ggt tta gag aac ggg gat tac aca 2688Ser Asn Gly Lys Tyr Glu Phe Thr Gly Leu Glu Asn Gly Asp Tyr Thr 885 890895 ata gaa ttt gag acg ccg gaa ggc tac aca ccg act aaa caa aac tcg 2736Ile Glu Phe Glu Thr Pro Glu Gly Tyr Thr Pro Thr Lys Gln Asn Ser 900 905910 gga agt gac gaa ggt aaa gat tca aac ggt acg aaa aca aca gtc aca 2784Gly Ser Asp Glu Gly Lys Asp Ser Asn Gly Thr Lys Thr Thr Val Thr 915 920925 gtc aaa gat gca gat aat aaa aca ata gac tca ggt ttc tac aag cca 2832Val Lys Asp Ala Asp Asn Lys Thr Ile Asp Ser Gly Phe Tyr Lys Pro 930 935940 aca tat aac tta ggt gac tat gta tgg gaa gat aca aat aaa gat ggt 2880Thr Tyr Asn Leu Gly Asp Tyr Val Trp Glu Asp Thr Asn Lys Asp Gly 945 950955 960 att caa gac gac agt gaa aaa ggg att tct ggg gtt aaa gtg acg tta2928 Ile Gln Asp Asp Ser Glu Lys Gly Ile Ser Gly Val Lys Val Thr Leu 965970 975 aaa gat aaa aat gga aat gcc att ggg aca acg aca aca gac gca agt2976 Lys Asp Lys Asn Gly Asn Ala Ile Gly Thr Thr Thr Thr Asp Ala Ser 980985 990 ggt cat tat caa ttt aaa gga tta gaa aat gga agc tac aca gtt gag3024 Gly His Tyr Gln Phe Lys Gly Leu Glu Asn Gly Ser Tyr Thr Val Glu 9951000 1005 ttt gag aca cca tca ggt tat aca ccg aca aaa gcg aat tca ggtcaa 3072 Phe Glu Thr Pro Ser Gly Tyr Thr Pro Thr Lys Ala Asn Ser Gly Gln1010 1015 1020 gat ata act gta gat tcc aac ggt ata aca aca aca ggt atcatt aac 3120 Asp Ile Thr Val Asp Ser Asn Gly Ile Thr Thr Thr Gly Ile IleAsn 1025 1030 1035 1040 gga gct gat aat ctc aca att gat agt ggt ttc tacaaa aca cca aaa 3168 Gly Ala Asp Asn Leu Thr Ile Asp Ser Gly Phe Tyr LysThr Pro Lys 1045 1050 1055 tat agt gtc gga gat tat gta tgg gaa gat acaaat aaa gat ggt atc 3216 Tyr Ser Val Gly Asp Tyr Val Trp Glu Asp Thr AsnLys Asp Gly Ile 1060 1065 1070 caa gat gac aat gaa aag gga att tct ggtgtt aaa gta acg tta aag 3264 Gln Asp Asp Asn Glu Lys Gly Ile Ser Gly ValLys Val Thr Leu Lys 1075 1080 1085 gat gaa aaa gga aat ata att agc actaca aca act gat gaa aat ggg 3312 Asp Glu Lys Gly Asn Ile Ile Ser Thr ThrThr Thr Asp Glu Asn Gly 1090 1095 1100 aag tat caa ttt gat aat tta gatagt ggt aat tac att att cat ttt 3360 Lys Tyr Gln Phe Asp Asn Leu Asp SerGly Asn Tyr Ile Ile His Phe 1105 1110 1115 1120 gag aaa ccg gaa ggc atgact caa act aca gca aat tct gga aat gat 3408 Glu Lys Pro Glu Gly Met ThrGln Thr Thr Ala Asn Ser Gly Asn Asp 1125 1130 1135 gat gaa aaa gat gctgat ggg gaa gat gtt cgt gtt acg att act gat 3456 Asp Glu Lys Asp Ala AspGly Glu Asp Val Arg Val Thr Ile Thr Asp 1140 1145 1150 cat gat gac tttagt ata gat aat ggt tat ttt gac gat gat tca gac 3504 His Asp Asp Phe SerIle Asp Asn Gly Tyr Phe Asp Asp Asp Ser Asp 1155 1160 1165 agt gac tcagac gca gat agt gat tca gac tca gac agt gac tcg gac 3552 Ser Asp Ser AspAla Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1170 1175 1180 gca gacagc gat tct gac gca gac agt gac tca gac gca gat agt gat 3600 Ala Asp SerAsp Ser Asp Ala Asp Ser Asp Ser Asp Ala Asp Ser Asp 1185 1190 1195 1200tct gac tca gac agc gac tca gac gca gat agt gat tcc gat tca gac 3648 SerAsp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ser Asp 1205 12101215 agc gac tcg gat tca gat agt gat tcg gat gca gac agc gac tcg gat3696 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp1220 1225 1230 tct gac agt gat tct gac gca gac agt gac tca gat tca gacagt gac 3744 Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ser Asp SerAsp 1235 1240 1245 tcg gat tca gac agc gat tcg gat tcc gat tca gac agtgac tcg gat 3792 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp 1250 1255 1260 tca gac agt gac tca gac tcc gac agt gat tcc gattca gat agc gac 3840 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp 1265 1270 1275 1280 tcc gac gca gat agt gat tcg gac gca gacagt gac tca gat tca gac 3888 Ser Asp Ala Asp Ser Asp Ser Asp Ala Asp SerAsp Ser Asp Ser Asp 1285 1290 1295 agt gat tcg gac gca gac agt gac tcggac tca gat agt gat tca gat 3936 Ser Asp Ser Asp Ala Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp 1300 1305 1310 gca gac agc gat tca gac tca gatagc gac tcg gat tca gac agc gac 3984 Ala Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp 1315 1320 1325 tcc gac gca gac agc gac tcggat tca gat agt gat tct gac tca gac 4032 Ser Asp Ala Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 1330 1335 1340 agt gac tca gat tcc gatagt gat tcg gat tca gat agt gat tcc gac 4080 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 1345 1350 1355 1360 gca gac agc gattcg gat tcc gat agc gat tca gac tca gac agc gat 4128 Ala Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1365 1370 1375 tca gat tcagac agc gac tca gat tca gat agt gat tcc gac gca gac 4176 Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp 1380 1385 1390 agc gatgca gac agc gac tca gac gca gac agt gat tca gat gca gac 4224 Ser Asp AlaAsp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ala Asp 1395 1400 1405 agcgat tct gac tca gat agt gac tca gac gca gat agt gat tcc gat 4272 Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp 1410 1415 1420tcc gat agc gat tca gat tct gat agt gac tca gac tca gac agt gac 4320 SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1425 14301435 1440 tca gat tcc gat agc gac tcg gat tca gat agt gat tcc gac gcagac 4368 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp1445 1450 1455 agt gac tca gac tca gat agt gac tcg gat tcc gat agt gattcc gac 4416 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp 1460 1465 1470 gca gac agc gat tct gac tca gat agt gac tca gac gcagat agt gat 4464 Ala Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala AspSer Asp 1475 1480 1485 tcc gat tcc gat agc gat tcg gat gca gac agc gactcg gat tca gat 4512 Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp SerAsp Ser Asp 1490 1495 1500 agt gat tcc gac gca gac agt gac tca gac tcagat agt gac tcg gat 4560 Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp 1505 1510 1515 1520 tcc gat agt gat tcc gac gca gac agcgat tcg gat tcc gat agc gat 4608 Ser Asp Ser Asp Ser Asp Ala Asp Ser AspSer Asp Ser Asp Ser Asp 1525 1530 1535 tca gac tcc gac agc gat tca gattca gac agc gac tca gat tcc gat 4656 Ser Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp 1540 1545 1550 agt gat tcc gat tca gac agtgac tcg gat tcc gat agt gac tca gac 4704 Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 1555 1560 1565 tca gac agt gac tca gattca gat agc gac tca gat tca gac agt gat 4752 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 1570 1575 1580 tcg gac tca gat agtgac tcc gat tca gac agt gat tcg gat tcc gat 4800 Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp 1585 1590 1595 1600 agc gat tcggat tcc gat agt gac tcg gat tca gac agt gat tcg gac 4848 Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1605 1610 1615 tca gacagc gac tcc gat tca gat agt gat tcc gac tca gac agc gat 4896 Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1620 1625 1630 tcggat tcc gat agt gac tcg gat tca gac agt gat tcg gac tca gac 4944 Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1635 1640 1645agc gac tcc gat tca gat agt gat tcc gac gca gac agc gac tcc gat 4992 SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp 1650 16551660 tca gat agt gat tcg gac gca gac agc gat tcc gat agt gac tcg gat5040 Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ser Asp Ser Asp1665 1670 1675 1680 tca gac agt gat tcg gac tca gac agc gat tcc gat tcagac agt gac 5088 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp 1685 1690 1695 tcg gac tca gat agc gac tcg gat tca gac agt gactcg gac tca gat 5136 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp 1700 1705 1710 agt gac tcc gat tca gac agc gac tcg gat tctgat aaa aat gca aaa 5184 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspLys Asn Ala Lys 1715 1720 1725 gat aaa tta cct gat aca gga gca aat gaagat cat gat tct aaa ggc 5232 Asp Lys Leu Pro Asp Thr Gly Ala Asn Glu AspHis Asp Ser Lys Gly 1730 1735 1740 aca tta ctt gga act tta ttt gca ggttta gga gca tta tta tta gga 5280 Thr Leu Leu Gly Thr Leu Phe Ala Gly LeuGly Ala Leu Leu Leu Gly 1745 1750 1755 1760 aga cgt cgt aaa aaa gat aataaa gaa aaa tag cac tat tga ttc att 5328 Arg Arg Arg Lys Lys Asp Asn LysGlu Lys His Tyr Phe Ile 1765 1770 1775 cat aag tta ttt caa gcc agg tctata tgg cct ggt ttg aaa tca tat 5376 His Lys Leu Phe Gln Ala Arg Ser IleTrp Pro Gly Leu Lys Ser Tyr 1780 1785 1790 taa att gaa agg aga aaa agatga gta tgg 5406 Ile Glu Arg Arg Lys Arg Val Trp 1795 1800 2 11 PRTStaphylococcus epidermidis 2 Tyr Trp Ile Asn Tyr Ala Tyr Lys Val Phe Thr1 5 10 3 15 PRT Staphylococcus epidermidis 3 Lys Cys Lys Cys Asn Leu GlnVal Asn Ile Gln Ile Ile Ser Leu 1 5 10 15 4 1742 PRT Staphylococcusepidermidis 4 Asn Ile Tyr Phe Asn Trp Arg Tyr Ser Met Lys Lys Arg ArgGln Gly 1 5 10 15 Pro Ile Asn Lys Arg Val Asp Phe Leu Ser Asn Lys ValAsn Lys Tyr 20 25 30 Ser Ile Arg Lys Phe Thr Val Gly Thr Ala Ser Ile LeuVal Gly Ala 35 40 45 Thr Leu Met Phe Gly Ala Ala Asp Asn Glu Ala Lys AlaAla Glu Asp 50 55 60 Asn Gln Leu Glu Ser Ala Ser Lys Glu Glu Gln Lys GlySer Arg Asp 65 70 75 80 Asn Glu Asn Ser Lys Leu Asn Gln Val Asp Leu AspAsn Gly Ser His 85 90 95 Ser Ser Glu Lys Thr Thr Asn Val Asn Asn Ala ThrGlu Val Lys Lys 100 105 110 Val Glu Ala Pro Thr Thr Ser Asp Val Ser LysPro Lys Ala Asn Glu 115 120 125 Ala Val Val Thr Asn Glu Ser Thr Lys ProLys Thr Thr Glu Ala Pro 130 135 140 Thr Val Asn Glu Glu Ser Ile Ala GluThr Pro Lys Thr Ser Thr Thr 145 150 155 160 Gln Gln Asp Ser Thr Glu LysAsn Asn Pro Ser Leu Lys Asp Asn Leu 165 170 175 Asn Ser Ser Ser Thr ThrSer Lys Glu Ser Lys Thr Asp Glu His Ser 180 185 190 Thr Lys Gln Ala GlnMet Ser Thr Asn Lys Ser Asn Leu Asp Thr Asn 195 200 205 Asp Ser Pro ThrGln Ser Glu Lys Thr Ser Ser Gln Ala Asn Asn Asp 210 215 220 Ser Thr AspAsn Gln Ser Ala Pro Ser Lys Gln Leu Asp Ser Lys Pro 225 230 235 240 SerGlu Gln Lys Val Tyr Lys Thr Lys Phe Asn Asp Glu Pro Thr Gln 245 250 255Asp Val Glu His Thr Thr Thr Lys Leu Lys Thr Pro Ser Val Ser Thr 260 265270 Asp Ser Ser Val Asn Asp Lys Gln Asp Tyr Thr Arg Ser Ala Val Ala 275280 285 Ser Leu Gly Val Asp Ser Asn Glu Thr Glu Ala Ile Thr Asn Ala Val290 295 300 Arg Asp Asn Leu Asp Leu Lys Ala Ala Ser Arg Glu Gln Ile AsnGlu 305 310 315 320 Ala Ile Ile Ala Glu Ala Leu Lys Lys Asp Phe Ser AsnPro Asp Tyr 325 330 335 Gly Val Asp Thr Pro Leu Ala Leu Asn Arg Ser GlnSer Lys Asn Ser 340 345 350 Pro His Lys Ser Ala Ser Pro Arg Met Asn LeuMet Ser Leu Ala Ala 355 360 365 Glu Pro Asn Ser Gly Lys Asn Val Asn AspLys Val Lys Ile Thr Asn 370 375 380 Pro Thr Leu Ser Leu Asn Lys Ser AsnAsn His Ala Asn Asn Val Ile 385 390 395 400 Trp Pro Thr Ser Asn Glu GlnPhe Asn Leu Lys Ala Asn Tyr Glu Leu 405 410 415 Asp Asp Ser Ile Lys GluGly Asp Thr Phe Thr Ile Lys Tyr Gly Gln 420 425 430 Tyr Ile Arg Pro GlyGly Leu Glu Leu Pro Ala Ile Lys Thr Gln Leu 435 440 445 Arg Ser Lys AspGly Ser Ile Val Ala Asn Gly Val Tyr Asp Lys Thr 450 455 460 Thr Asn ThrThr Thr Tyr Thr Phe Thr Asn Tyr Val Asp Gln Tyr Gln 465 470 475 480 AsnIle Thr Gly Ser Phe Asp Leu Ile Ala Thr Pro Lys Arg Glu Thr 485 490 495Ala Ile Lys Asp Asn Gln Asn Tyr Pro Met Glu Val Thr Ile Ala Asn 500 505510 Glu Val Val Lys Lys Asp Phe Ile Val Asp Tyr Gly Asn Lys Lys Asp 515520 525 Asn Thr Thr Thr Ala Ala Val Ala Asn Val Asp Asn Val Asn Asn Lys530 535 540 His Asn Glu Val Val Tyr Leu Asn Gln Asn Asn Gln Asn Pro LysTyr 545 550 555 560 Ala Lys Tyr Phe Ser Thr Val Lys Asn Gly Glu Phe IlePro Gly Glu 565 570 575 Val Lys Val Tyr Glu Val Thr Asp Thr Asn Ala MetVal Asp Ser Phe 580 585 590 Asn Pro Asp Leu Asn Ser Ser Asn Val Lys AspVal Thr Ser Gln Phe 595 600 605 Ala Pro Lys Val Ser Ala Asp Gly Thr ArgVal Asp Ile Asn Phe Ala 610 615 620 Arg Ser Met Ala Asn Gly Lys Lys TyrIle Val Thr Gln Ala Val Arg 625 630 635 640 Pro Thr Gly Thr Gly Asn ValTyr Thr Glu Tyr Trp Leu Thr Arg Asp 645 650 655 Gly Thr Thr Asn Thr AsnAsp Phe Tyr Arg Gly Thr Lys Ser Thr Thr 660 665 670 Val Thr Tyr Leu AsnGly Ser Ser Thr Ala Gln Gly Asp Asn Pro Thr 675 680 685 Tyr Ser Leu GlyAsp Tyr Val Trp Leu Asp Lys Asn Lys Asn Gly Val 690 695 700 Gln Asp AspAsp Glu Lys Gly Leu Ala Gly Val Tyr Val Thr Leu Lys 705 710 715 720 AspSer Asn Asn Arg Glu Leu Gln Arg Val Thr Thr Asp Gln Ser Gly 725 730 735His Tyr Gln Phe Asp Asn Leu Gln Asn Gly Thr Tyr Thr Val Glu Phe 740 745750 Ala Ile Pro Asp Asn Tyr Thr Pro Ser Pro Ala Asn Asn Ser Thr Asn 755760 765 Asp Ala Ile Asp Ser Asp Gly Glu Arg Asp Gly Thr Arg Lys Val Val770 775 780 Val Ala Lys Gly Thr Ile Asn Asn Ala Asp Asn Met Thr Val AspThr 785 790 795 800 Gly Phe Tyr Leu Thr Pro Lys Tyr Asn Val Gly Asp TyrVal Trp Glu 805 810 815 Asp Thr Asn Lys Asp Gly Ile Gln Asp Asp Asn GluLys Gly Ile Ser 820 825 830 Gly Val Lys Val Thr Leu Lys Asn Lys Asn GlyAsp Thr Ile Gly Thr 835 840 845 Thr Thr Thr Asp Ser Asn Gly Lys Tyr GluPhe Thr Gly Leu Glu Asn 850 855 860 Gly Asp Tyr Thr Ile Glu Phe Glu ThrPro Glu Gly Tyr Thr Pro Thr 865 870 875 880 Lys Gln Asn Ser Gly Ser AspGlu Gly Lys Asp Ser Asn Gly Thr Lys 885 890 895 Thr Thr Val Thr Val LysAsp Ala Asp Asn Lys Thr Ile Asp Ser Gly 900 905 910 Phe Tyr Lys Pro ThrTyr Asn Leu Gly Asp Tyr Val Trp Glu Asp Thr 915 920 925 Asn Lys Asp GlyIle Gln Asp Asp Ser Glu Lys Gly Ile Ser Gly Val 930 935 940 Lys Val ThrLeu Lys Asp Lys Asn Gly Asn Ala Ile Gly Thr Thr Thr 945 950 955 960 ThrAsp Ala Ser Gly His Tyr Gln Phe Lys Gly Leu Glu Asn Gly Ser 965 970 975Tyr Thr Val Glu Phe Glu Thr Pro Ser Gly Tyr Thr Pro Thr Lys Ala 980 985990 Asn Ser Gly Gln Asp Ile Thr Val Asp Ser Asn Gly Ile Thr Thr Thr 9951000 1005 Gly Ile Ile Asn Gly Ala Asp Asn Leu Thr Ile Asp Ser Gly PheTyr 1010 1015 1020 Lys Thr Pro Lys Tyr Ser Val Gly Asp Tyr Val Trp GluAsp Thr Asn 025 1030 1035 1040 Lys Asp Gly Ile Gln Asp Asp Asn Glu LysGly Ile Ser Gly Val Lys 1045 1050 1055 Val Thr Leu Lys Asp Glu Lys GlyAsn Ile Ile Ser Thr Thr Thr Thr 1060 1065 1070 Asp Glu Asn Gly Lys TyrGln Phe Asp Asn Leu Asp Ser Gly Asn Tyr 1075 1080 1085 Ile Ile His PheGlu Lys Pro Glu Gly Met Thr Gln Thr Thr Ala Asn 1090 1095 1100 Ser GlyAsn Asp Asp Glu Lys Asp Ala Asp Gly Glu Asp Val Arg Val 105 1110 11151120 Thr Ile Thr Asp His Asp Asp Phe Ser Ile Asp Asn Gly Tyr Phe Asp1125 1130 1135 Asp Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser AspSer Asp 1140 1145 1150 Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ala AspSer Asp Ser Asp 1155 1160 1165 Ala Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ala Asp Ser Asp 1170 1175 1180 Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ala Asp 185 1190 1195 1200 Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp 1205 1210 1215 Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1220 1225 1230 SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1235 12401245 Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ala Asp Ser Asp1250 1255 1260 Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser AspSer Asp 265 1270 1275 1280 Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp 1285 1290 1295 Ser Asp Ser Asp Ser Asp Ala Asp SerAsp Ser Asp Ser Asp Ser Asp 1300 1305 1310 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 1315 1320 1325 Ser Asp Ser Asp AlaAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1330 1335 1340 Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 345 1350 1355 1360Ser Asp Ala Asp Ser Asp Ala Asp Ser Asp Ser Asp Ala Asp Ser Asp 13651370 1375 Ser Asp Ala Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp AlaAsp 1380 1385 1390 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp 1395 1400 1405 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp 1410 1415 1420 Ser Asp Ala Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp 425 1430 1435 1440 Ser Asp Ser Asp Ala AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp 1445 1450 1455 Ala Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp 1460 1465 1470 Ser AspSer Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp Ser Asp 1475 1480 1485Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser Asp Ser Asp 14901495 1500 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp SerAsp 505 1510 1515 1520 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp 1525 1530 1535 Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp 1540 1545 1550 Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 1555 1560 1565 Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp 1570 1575 1580 Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 585 1590 1595 1600 SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp 1605 16101615 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp1620 1625 1630 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ala Asp Ser AspSer Asp 1635 1640 1645 Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp 1650 1655 1660 Ser Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp 665 1670 1675 1680 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 1685 1690 1695 Lys Asn Ala Lys AspLys Leu Pro Asp Thr Gly Ala Asn Glu Asp His 1700 1705 1710 Asp Ser LysGly Thr Leu Leu Gly Thr Leu Phe Ala Gly Leu Gly Ala 1715 1720 1725 LeuLeu Leu Gly Arg Arg Arg Lys Lys Asp Asn Lys Glu Lys 1730 1735 1740 5 18PRT Staphylococcus epidermidis 5 Phe Ile His Lys Leu Phe Gln Ala Arg SerIle Trp Pro Gly Leu Lys 1 5 10 15 Ser Tyr 6 6 PRT Staphylococcusepidermidis 6 Ile Glu Arg Arg Lys Arg 1 5 7 2976 DNA Staphylococcusepidermidis CDS (3)..(2975) 7 at att gca aaa aag act tat ata cta tat tgtatt tta ctc tag aaa 47 Ile Ala Lys Lys Thr Tyr Ile Leu Tyr Cys Ile LeuLeu Lys 1 5 10 15 cga ttt tta ctt gaa aat tac att gaa ata gtc aaa gataag gag ttt 95 Arg Phe Leu Leu Glu Asn Tyr Ile Glu Ile Val Lys Asp LysGlu Phe 20 25 30 tta tga tta aaa aaa aat aat tta cta act aaa aag aaa cctata gca 143 Leu Leu Lys Lys Asn Asn Leu Leu Thr Lys Lys Lys Pro Ile Ala35 40 45 aat aaa tcc aat aaa tat gca att aga aaa ttc aca gta ggt aca gcg191 Asn Lys Ser Asn Lys Tyr Ala Ile Arg Lys Phe Thr Val Gly Thr Ala 5055 60 tct att gta ata ggt gca gca tta ttg ttt ggt tta ggt cat aat gag239 Ser Ile Val Ile Gly Ala Ala Leu Leu Phe Gly Leu Gly His Asn Glu 6570 75 gcc aaa gct gag gag aat aca gta caa gac gtt aaa gat tcg aat atg287 Ala Lys Ala Glu Glu Asn Thr Val Gln Asp Val Lys Asp Ser Asn Met 8085 90 95 gat gat gaa tta tca gat agc aat gat cag tcc agt aat gaa gaa aag335 Asp Asp Glu Leu Ser Asp Ser Asn Asp Gln Ser Ser Asn Glu Glu Lys 100105 110 aat gat gta atc aat aat agt cag tca ata aac acc gat gat gat aac383 Asn Asp Val Ile Asn Asn Ser Gln Ser Ile Asn Thr Asp Asp Asp Asn 115120 125 caa ata aaa aaa gaa gaa acg aat agc aac gat gcc ata gaa aat cgc431 Gln Ile Lys Lys Glu Glu Thr Asn Ser Asn Asp Ala Ile Glu Asn Arg 130135 140 tct aaa gat ata aca cag tca aca aca aat gta gat gaa aac gaa gca479 Ser Lys Asp Ile Thr Gln Ser Thr Thr Asn Val Asp Glu Asn Glu Ala 145150 155 aca ttt tta caa aag acc cct caa gat aat act cag ctt aaa gaa gaa527 Thr Phe Leu Gln Lys Thr Pro Gln Asp Asn Thr Gln Leu Lys Glu Glu 160165 170 175 gtg gta aaa gaa ccc tca tca gtc gaa tcc tca aat tca tca atggat 575 Val Val Lys Glu Pro Ser Ser Val Glu Ser Ser Asn Ser Ser Met Asp180 185 190 act gcc caa caa cca tct cat aca aca ata aat agt gaa gca tctatt 623 Thr Ala Gln Gln Pro Ser His Thr Thr Ile Asn Ser Glu Ala Ser Ile195 200 205 caa aca agt gat aat gaa gaa aat tcc cgc gta tca gat ttt gctaac 671 Gln Thr Ser Asp Asn Glu Glu Asn Ser Arg Val Ser Asp Phe Ala Asn210 215 220 tct aaa ata ata gag agt aac act gaa tcc aat aaa gaa gag aatact 719 Ser Lys Ile Ile Glu Ser Asn Thr Glu Ser Asn Lys Glu Glu Asn Thr225 230 235 ata gag caa cct aac aaa gta aga gaa gat tca ata aca agt caaccg 767 Ile Glu Gln Pro Asn Lys Val Arg Glu Asp Ser Ile Thr Ser Gln Pro240 245 250 255 tct agc tat aaa aat ata gat gaa aaa att tca aat caa gatgag tta 815 Ser Ser Tyr Lys Asn Ile Asp Glu Lys Ile Ser Asn Gln Asp GluLeu 260 265 270 tta aat tta cca ata aat gaa tat gaa aat aag gtt aga ccgtta tct 863 Leu Asn Leu Pro Ile Asn Glu Tyr Glu Asn Lys Val Arg Pro LeuSer 275 280 285 aca aca tct gcc caa cca tcg agt aag cgt gta acc gta aatcaa tta 911 Thr Thr Ser Ala Gln Pro Ser Ser Lys Arg Val Thr Val Asn GlnLeu 290 295 300 gcg gca gaa caa ggt tcg aat gtt aat cat tta att aaa gttact gat 959 Ala Ala Glu Gln Gly Ser Asn Val Asn His Leu Ile Lys Val ThrAsp 305 310 315 caa agt att act gaa gga tat gat gat agt gat ggt att attaaa gca 1007 Gln Ser Ile Thr Glu Gly Tyr Asp Asp Ser Asp Gly Ile Ile LysAla 320 325 330 335 cat gat gct gaa aac tta atc tat gat gta act ttt gaagta gat gat 1055 His Asp Ala Glu Asn Leu Ile Tyr Asp Val Thr Phe Glu ValAsp Asp 340 345 350 aag gtg aaa tct ggt gat acg atg aca gtg aat ata gataag aat aca 1103 Lys Val Lys Ser Gly Asp Thr Met Thr Val Asn Ile Asp LysAsn Thr 355 360 365 gtt cca tca gat tta acc gat agt ttt gca ata cca aaaata aaa gat 1151 Val Pro Ser Asp Leu Thr Asp Ser Phe Ala Ile Pro Lys IleLys Asp 370 375 380 aat tct gga gaa atc atc gct aca ggt act tat gac aacaca aat aaa 1199 Asn Ser Gly Glu Ile Ile Ala Thr Gly Thr Tyr Asp Asn ThrAsn Lys 385 390 395 caa att acc tac act ttt aca gat tat gta gat aaa tatgaa aat att 1247 Gln Ile Thr Tyr Thr Phe Thr Asp Tyr Val Asp Lys Tyr GluAsn Ile 400 405 410 415 aaa gcg cac ctt aaa tta aca tca tac att gat aaatca aag gtt cca 1295 Lys Ala His Leu Lys Leu Thr Ser Tyr Ile Asp Lys SerLys Val Pro 420 425 430 aat aat aac act aag tta gat gta gaa tat aag acggcc ctt tca tca 1343 Asn Asn Asn Thr Lys Leu Asp Val Glu Tyr Lys Thr AlaLeu Ser Ser 435 440 445 gta aat aaa aca att acg gtt gaa tat caa aaa cctaac gaa aat cgg 1391 Val Asn Lys Thr Ile Thr Val Glu Tyr Gln Lys Pro AsnGlu Asn Arg 450 455 460 act gct aac ctt caa agt atg ttc aca aac ata gatacg aaa aac cat 1439 Thr Ala Asn Leu Gln Ser Met Phe Thr Asn Ile Asp ThrLys Asn His 465 470 475 aca gtt gag caa acg att tat att aac cct ctt cgttat tca gcc aaa 1487 Thr Val Glu Gln Thr Ile Tyr Ile Asn Pro Leu Arg TyrSer Ala Lys 480 485 490 495 gaa aca aat gta aat att tca ggg aat ggc gatgaa ggt tca aca att 1535 Glu Thr Asn Val Asn Ile Ser Gly Asn Gly Asp GluGly Ser Thr Ile 500 505 510 atc gac gat agt aca atc att aaa gtt tat aaggtt gga gat aat caa 1583 Ile Asp Asp Ser Thr Ile Ile Lys Val Tyr Lys ValGly Asp Asn Gln 515 520 525 aat tta cca gat agt aac aga att tat gat tacagt gaa tat gaa gat 1631 Asn Leu Pro Asp Ser Asn Arg Ile Tyr Asp Tyr SerGlu Tyr Glu Asp 530 535 540 gtc aca aat gat gat tat gcc caa tta gga aataat aat gac gtg aat 1679 Val Thr Asn Asp Asp Tyr Ala Gln Leu Gly Asn AsnAsn Asp Val Asn 545 550 555 att aat ttt ggt aat ata gat tca cca tat attatt aaa gtt att agt 1727 Ile Asn Phe Gly Asn Ile Asp Ser Pro Tyr Ile IleLys Val Ile Ser 560 565 570 575 aaa tat gac cct aat aag gac gat tac acgacg ata cag caa act gtg 1775 Lys Tyr Asp Pro Asn Lys Asp Asp Tyr Thr ThrIle Gln Gln Thr Val 580 585 590 aca atg caa acg act ata aat gag tat actggt gag ttt aga aca gca 1823 Thr Met Gln Thr Thr Ile Asn Glu Tyr Thr GlyGlu Phe Arg Thr Ala 595 600 605 tcc tat gat aat aca att gct ttc tct acaagt tca ggt caa gga caa 1871 Ser Tyr Asp Asn Thr Ile Ala Phe Ser Thr SerSer Gly Gln Gly Gln 610 615 620 ggt gac ttg cct cct gaa aaa act tat aaaatc gga gat tac gta tgg 1919 Gly Asp Leu Pro Pro Glu Lys Thr Tyr Lys IleGly Asp Tyr Val Trp 625 630 635 gaa gat gta gat aaa gat ggt att caa aataca aat gat aat gaa aaa 1967 Glu Asp Val Asp Lys Asp Gly Ile Gln Asn ThrAsn Asp Asn Glu Lys 640 645 650 655 ccg ctt agt aat gta ttg gta act ttgacg tat cct gat gga act tca 2015 Pro Leu Ser Asn Val Leu Val Thr Leu ThrTyr Pro Asp Gly Thr Ser 660 665 670 aaa tca gtc aga aca gat gaa gag gggaaa tat caa ttt gat ggg tta 2063 Lys Ser Val Arg Thr Asp Glu Glu Gly LysTyr Gln Phe Asp Gly Leu 675 680 685 aaa aac gga ttg act tat aaa att acattc gaa aca ccg gaa gga tat 2111 Lys Asn Gly Leu Thr Tyr Lys Ile Thr PheGlu Thr Pro Glu Gly Tyr 690 695 700 acg ccg acg ctt aaa cat tca gga acaaat cct gca cta gac tca gaa 2159 Thr Pro Thr Leu Lys His Ser Gly Thr AsnPro Ala Leu Asp Ser Glu 705 710 715 ggc aat tct gta tgg gta act att aacgga caa gac gat atg act att 2207 Gly Asn Ser Val Trp Val Thr Ile Asn GlyGln Asp Asp Met Thr Ile 720 725 730 735 gat agc gga ttt tat caa aca cctaaa tat agc tta ggg aac tat gta 2255 Asp Ser Gly Phe Tyr Gln Thr Pro LysTyr Ser Leu Gly Asn Tyr Val 740 745 750 tgg tat gac act aat aaa gat ggtatt caa ggt gat gat gaa aaa gga 2303 Trp Tyr Asp Thr Asn Lys Asp Gly IleGln Gly Asp Asp Glu Lys Gly 755 760 765 atc tct gga gta aaa gtg acg ttaaaa gat gaa aac gga aat atc att 2351 Ile Ser Gly Val Lys Val Thr Leu LysAsp Glu Asn Gly Asn Ile Ile 770 775 780 agt aca aca aca act gat gaa aatgga aag tat caa ttt gat aat tta 2399 Ser Thr Thr Thr Thr Asp Glu Asn GlyLys Tyr Gln Phe Asp Asn Leu 785 790 795 aat agt ggt aat tat att gtt catttt gat aaa cct tca ggt atg act 2447 Asn Ser Gly Asn Tyr Ile Val His PheAsp Lys Pro Ser Gly Met Thr 800 805 810 815 caa aca aca aca gat tct ggtgat gat gac gaa cag gat gct gat ggg 2495 Gln Thr Thr Thr Asp Ser Gly AspAsp Asp Glu Gln Asp Ala Asp Gly 820 825 830 gaa gaa gtc cat gta aca attact gat cat gat gac ttt agt ata gat 2543 Glu Glu Val His Val Thr Ile ThrAsp His Asp Asp Phe Ser Ile Asp 835 840 845 aac gga tac tat gat gac gactca gat tca gat agt gat tca gac tca 2591 Asn Gly Tyr Tyr Asp Asp Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 850 855 860 gat agc gac gac tca gac tccgat agc gat tcc gac tca gac agc gac 2639 Asp Ser Asp Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 865 870 875 tca gat tcc gat agt gat tcagat tca gac agt gac tca gac tca gat 2687 Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp 880 885 890 895 agt gat tca gat tca gacagc gat tcc gac tca gac agt gac tca gga 2735 Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Gly 900 905 910 tta gac aat agc tca gataag aat aca aaa gat aaa tta ccg gat aca 2783 Leu Asp Asn Ser Ser Asp LysAsn Thr Lys Asp Lys Leu Pro Asp Thr 915 920 925 gga gct aat gaa gat catgat tct aaa ggc aca tta ctt gga gct tta 2831 Gly Ala Asn Glu Asp His AspSer Lys Gly Thr Leu Leu Gly Ala Leu 930 935 940 ttt gca ggt tta gga gcgtta tta tta ggg aag cgt cgc aaa aat aga 2879 Phe Ala Gly Leu Gly Ala LeuLeu Leu Gly Lys Arg Arg Lys Asn Arg 945 950 955 aaa aat aaa aat taa attatt caa atg aaa tta gtg aaa gaa gca gat 2927 Lys Asn Lys Asn Ile Ile GlnMet Lys Leu Val Lys Glu Ala Asp 960 965 970 975 acg aca ttt gaa tag aaagta tat tta gtc caa caa ata taa ggt gtt g 2976 Thr Thr Phe Glu Lys ValTyr Leu Val Gln Gln Ile Gly Val 980 985 990 8 13 PRT Staphylococcusepidermidis 8 Ile Ala Lys Lys Thr Tyr Ile Leu Tyr Cys Ile Leu Leu 1 5 109 18 PRT Staphylococcus epidermidis 9 Lys Arg Phe Leu Leu Glu Asn TyrIle Glu Ile Val Lys Asp Lys Glu 1 5 10 15 Phe Leu 10 930 PRTStaphylococcus epidermidis 10 Leu Lys Lys Asn Asn Leu Leu Thr Lys LysLys Pro Ile Ala Asn Lys 1 5 10 15 Ser Asn Lys Tyr Ala Ile Arg Lys PheThr Val Gly Thr Ala Ser Ile 20 25 30 Val Ile Gly Ala Ala Leu Leu Phe GlyLeu Gly His Asn Glu Ala Lys 35 40 45 Ala Glu Glu Asn Thr Val Gln Asp ValLys Asp Ser Asn Met Asp Asp 50 55 60 Glu Leu Ser Asp Ser Asn Asp Gln SerSer Asn Glu Glu Lys Asn Asp 65 70 75 80 Val Ile Asn Asn Ser Gln Ser IleAsn Thr Asp Asp Asp Asn Gln Ile 85 90 95 Lys Lys Glu Glu Thr Asn Ser AsnAsp Ala Ile Glu Asn Arg Ser Lys 100 105 110 Asp Ile Thr Gln Ser Thr ThrAsn Val Asp Glu Asn Glu Ala Thr Phe 115 120 125 Leu Gln Lys Thr Pro GlnAsp Asn Thr Gln Leu Lys Glu Glu Val Val 130 135 140 Lys Glu Pro Ser SerVal Glu Ser Ser Asn Ser Ser Met Asp Thr Ala 145 150 155 160 Gln Gln ProSer His Thr Thr Ile Asn Ser Glu Ala Ser Ile Gln Thr 165 170 175 Ser AspAsn Glu Glu Asn Ser Arg Val Ser Asp Phe Ala Asn Ser Lys 180 185 190 IleIle Glu Ser Asn Thr Glu Ser Asn Lys Glu Glu Asn Thr Ile Glu 195 200 205Gln Pro Asn Lys Val Arg Glu Asp Ser Ile Thr Ser Gln Pro Ser Ser 210 215220 Tyr Lys Asn Ile Asp Glu Lys Ile Ser Asn Gln Asp Glu Leu Leu Asn 225230 235 240 Leu Pro Ile Asn Glu Tyr Glu Asn Lys Val Arg Pro Leu Ser ThrThr 245 250 255 Ser Ala Gln Pro Ser Ser Lys Arg Val Thr Val Asn Gln LeuAla Ala 260 265 270 Glu Gln Gly Ser Asn Val Asn His Leu Ile Lys Val ThrAsp Gln Ser 275 280 285 Ile Thr Glu Gly Tyr Asp Asp Ser Asp Gly Ile IleLys Ala His Asp 290 295 300 Ala Glu Asn Leu Ile Tyr Asp Val Thr Phe GluVal Asp Asp Lys Val 305 310 315 320 Lys Ser Gly Asp Thr Met Thr Val AsnIle Asp Lys Asn Thr Val Pro 325 330 335 Ser Asp Leu Thr Asp Ser Phe AlaIle Pro Lys Ile Lys Asp Asn Ser 340 345 350 Gly Glu Ile Ile Ala Thr GlyThr Tyr Asp Asn Thr Asn Lys Gln Ile 355 360 365 Thr Tyr Thr Phe Thr AspTyr Val Asp Lys Tyr Glu Asn Ile Lys Ala 370 375 380 His Leu Lys Leu ThrSer Tyr Ile Asp Lys Ser Lys Val Pro Asn Asn 385 390 395 400 Asn Thr LysLeu Asp Val Glu Tyr Lys Thr Ala Leu Ser Ser Val Asn 405 410 415 Lys ThrIle Thr Val Glu Tyr Gln Lys Pro Asn Glu Asn Arg Thr Ala 420 425 430 AsnLeu Gln Ser Met Phe Thr Asn Ile Asp Thr Lys Asn His Thr Val 435 440 445Glu Gln Thr Ile Tyr Ile Asn Pro Leu Arg Tyr Ser Ala Lys Glu Thr 450 455460 Asn Val Asn Ile Ser Gly Asn Gly Asp Glu Gly Ser Thr Ile Ile Asp 465470 475 480 Asp Ser Thr Ile Ile Lys Val Tyr Lys Val Gly Asp Asn Gln AsnLeu 485 490 495 Pro Asp Ser Asn Arg Ile Tyr Asp Tyr Ser Glu Tyr Glu AspVal Thr 500 505 510 Asn Asp Asp Tyr Ala Gln Leu Gly Asn Asn Asn Asp ValAsn Ile Asn 515 520 525 Phe Gly Asn Ile Asp Ser Pro Tyr Ile Ile Lys ValIle Ser Lys Tyr 530 535 540 Asp Pro Asn Lys Asp Asp Tyr Thr Thr Ile GlnGln Thr Val Thr Met 545 550 555 560 Gln Thr Thr Ile Asn Glu Tyr Thr GlyGlu Phe Arg Thr Ala Ser Tyr 565 570 575 Asp Asn Thr Ile Ala Phe Ser ThrSer Ser Gly Gln Gly Gln Gly Asp 580 585 590 Leu Pro Pro Glu Lys Thr TyrLys Ile Gly Asp Tyr Val Trp Glu Asp 595 600 605 Val Asp Lys Asp Gly IleGln Asn Thr Asn Asp Asn Glu Lys Pro Leu 610 615 620 Ser Asn Val Leu ValThr Leu Thr Tyr Pro Asp Gly Thr Ser Lys Ser 625 630 635 640 Val Arg ThrAsp Glu Glu Gly Lys Tyr Gln Phe Asp Gly Leu Lys Asn 645 650 655 Gly LeuThr Tyr Lys Ile Thr Phe Glu Thr Pro Glu Gly Tyr Thr Pro 660 665 670 ThrLeu Lys His Ser Gly Thr Asn Pro Ala Leu Asp Ser Glu Gly Asn 675 680 685Ser Val Trp Val Thr Ile Asn Gly Gln Asp Asp Met Thr Ile Asp Ser 690 695700 Gly Phe Tyr Gln Thr Pro Lys Tyr Ser Leu Gly Asn Tyr Val Trp Tyr 705710 715 720 Asp Thr Asn Lys Asp Gly Ile Gln Gly Asp Asp Glu Lys Gly IleSer 725 730 735 Gly Val Lys Val Thr Leu Lys Asp Glu Asn Gly Asn Ile IleSer Thr 740 745 750 Thr Thr Thr Asp Glu Asn Gly Lys Tyr Gln Phe Asp AsnLeu Asn Ser 755 760 765 Gly Asn Tyr Ile Val His Phe Asp Lys Pro Ser GlyMet Thr Gln Thr 770 775 780 Thr Thr Asp Ser Gly Asp Asp Asp Glu Gln AspAla Asp Gly Glu Glu 785 790 795 800 Val His Val Thr Ile Thr Asp His AspAsp Phe Ser Ile Asp Asn Gly 805 810 815 Tyr Tyr Asp Asp Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 820 825 830 Asp Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp 835 840 845 Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp 850 855 860 Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Gly Leu Asp 865 870 875 880 Asn Ser SerAsp Lys Asn Thr Lys Asp Lys Leu Pro Asp Thr Gly Ala 885 890 895 Asn GluAsp His Asp Ser Lys Gly Thr Leu Leu Gly Ala Leu Phe Ala 900 905 910 GlyLeu Gly Ala Leu Leu Leu Gly Lys Arg Arg Lys Asn Arg Lys Asn 915 920 925Lys Asn 930 11 15 PRT Staphylococcus epidermidis 11 Ile Ile Gln Met LysLeu Val Lys Glu Ala Asp Thr Thr Phe Glu 1 5 10 15 12 8 PRTStaphylococcus epidermidis 12 Lys Val Tyr Leu Val Gln Gln Ile 1 5 131464 DNA Staphylococcus epidermidis CDS (1)..(1464) 13 atg aaa aag tttaac att aaa cat tca ttt atg ctt acg ggc ttt gct 48 Met Lys Lys Phe AsnIle Lys His Ser Phe Met Leu Thr Gly Phe Ala 1 5 10 15 ttc atg gta actaca tca tta ttc agt cac caa gca cat gct gaa ggt 96 Phe Met Val Thr ThrSer Leu Phe Ser His Gln Ala His Ala Glu Gly 20 25 30 aat cat cct att gacatt aat ttt tct aaa gat caa att gat aga aat 144 Asn His Pro Ile Asp IleAsn Phe Ser Lys Asp Gln Ile Asp Arg Asn 35 40 45 aca gct aag agc aat attatc aat cga gtg aat gac act agt cgc aca 192 Thr Ala Lys Ser Asn Ile IleAsn Arg Val Asn Asp Thr Ser Arg Thr 50 55 60 gga att agt atg aat tcg gataat gat tta gat aca gat atc gtt tca 240 Gly Ile Ser Met Asn Ser Asp AsnAsp Leu Asp Thr Asp Ile Val Ser 65 70 75 80 aat agt gac tca gaa aat gacaca tat tta gat agt gat tca gat tca 288 Asn Ser Asp Ser Glu Asn Asp ThrTyr Leu Asp Ser Asp Ser Asp Ser 85 90 95 gac agt gac tca gat tca gat agtgac tca gat tca gat agt gac tca 336 Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser 100 105 110 gat tca gat agt gac tca gat tcagac agt gat tca gac tca gat agt 384 Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser 115 120 125 gac tca gat tca gac agt gat tcagac tca gat agt gat tca gat tca 432 Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser 130 135 140 gac agt gat tca gat tca gac agtgac tca gac tca gac agt gat tca 480 Asp Ser Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser 145 150 155 160 gat tca gat agt gat tca gattca gat agt gat tca gat tca gat agt 528 Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 165 170 175 gat tca gat tca gac agt gactca gac tca gac agt gat tca gat tca 576 Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 180 185 190 gat agt gat tca gac tca gatagt gac tca gat tca gat agt gat tca 624 Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 195 200 205 gac tct ggt aca agt tca ggtaag ggt tca cat acc gga aaa aaa cct 672 Asp Ser Gly Thr Ser Ser Gly LysGly Ser His Thr Gly Lys Lys Pro 210 215 220 ggt aac cct aaa gga aat acaaat aga cct tct caa aga cat acg aat 720 Gly Asn Pro Lys Gly Asn Thr AsnArg Pro Ser Gln Arg His Thr Asn 225 230 235 240 caa ccc caa agg cct aaatac aat caa aca aat caa aac aat ata aac 768 Gln Pro Gln Arg Pro Lys TyrAsn Gln Thr Asn Gln Asn Asn Ile Asn 245 250 255 aat ata aac cat aat attaat cat aca cgt act agt gga gat ggt gcg 816 Asn Ile Asn His Asn Ile AsnHis Thr Arg Thr Ser Gly Asp Gly Ala 260 265 270 cct ttt aaa cgt caa caaaat att att aat tct aat tca ggt cat aga 864 Pro Phe Lys Arg Gln Gln AsnIle Ile Asn Ser Asn Ser Gly His Arg 275 280 285 aat caa aat aat ata aatcaa ttt ata tgg aac aaa aat ggc ttt ttt 912 Asn Gln Asn Asn Ile Asn GlnPhe Ile Trp Asn Lys Asn Gly Phe Phe 290 295 300 aaa tct caa aat aat accgaa cat aga atg aat agt agc gat aat acc 960 Lys Ser Gln Asn Asn Thr GluHis Arg Met Asn Ser Ser Asp Asn Thr 305 310 315 320 aat tca tta att agcaga ttc aga caa tta gcc acg ggt gct tat aag 1008 Asn Ser Leu Ile Ser ArgPhe Arg Gln Leu Ala Thr Gly Ala Tyr Lys 325 330 335 tac aat ccg ttt ttgatt aat caa gta aaa aat ttg aat caa tta gat 1056 Tyr Asn Pro Phe Leu IleAsn Gln Val Lys Asn Leu Asn Gln Leu Asp 340 345 350 gga aag gtg aca gatagt gac att tat agc ttg ttt aga aag caa tca 1104 Gly Lys Val Thr Asp SerAsp Ile Tyr Ser Leu Phe Arg Lys Gln Ser 355 360 365 ttt aga gga aat gaatat tta aat tca tta caa aaa ggg aca agc tat 1152 Phe Arg Gly Asn Glu TyrLeu Asn Ser Leu Gln Lys Gly Thr Ser Tyr 370 375 380 ttc aga ttt caa tatttt aat cca ctt aat tct agt aaa tac tat gaa 1200 Phe Arg Phe Gln Tyr PheAsn Pro Leu Asn Ser Ser Lys Tyr Tyr Glu 385 390 395 400 aat tta gat gatcag gtt tta gct tta att aca gga gaa atc ggc tca 1248 Asn Leu Asp Asp GlnVal Leu Ala Leu Ile Thr Gly Glu Ile Gly Ser 405 410 415 atg cca gaa cttaaa aaa cct acg gat aaa gaa gat aaa aat cat agc 1296 Met Pro Glu Leu LysLys Pro Thr Asp Lys Glu Asp Lys Asn His Ser 420 425 430 gcc ttc aaa aaccat agt gca gat gag ata aca aca aat aat gat gga 1344 Ala Phe Lys Asn HisSer Ala Asp Glu Ile Thr Thr Asn Asn Asp Gly 435 440 445 cac tcc aaa gattat gat aag aaa aag aaa ata cat cga agt ctt tta 1392 His Ser Lys Asp TyrAsp Lys Lys Lys Lys Ile His Arg Ser Leu Leu 450 455 460 tcg tta agt attgca ata att gga att ttt cta gga gtc act gga cta 1440 Ser Leu Ser Ile AlaIle Ile Gly Ile Phe Leu Gly Val Thr Gly Leu 465 470 475 480 tat atc tttaga aga aaa aag taa 1464 Tyr Ile Phe Arg Arg Lys Lys 485 14 487 PRTStaphylococcus epidermidis 14 Met Lys Lys Phe Asn Ile Lys His Ser PheMet Leu Thr Gly Phe Ala 1 5 10 15 Phe Met Val Thr Thr Ser Leu Phe SerHis Gln Ala His Ala Glu Gly 20 25 30 Asn His Pro Ile Asp Ile Asn Phe SerLys Asp Gln Ile Asp Arg Asn 35 40 45 Thr Ala Lys Ser Asn Ile Ile Asn ArgVal Asn Asp Thr Ser Arg Thr 50 55 60 Gly Ile Ser Met Asn Ser Asp Asn AspLeu Asp Thr Asp Ile Val Ser 65 70 75 80 Asn Ser Asp Ser Glu Asn Asp ThrTyr Leu Asp Ser Asp Ser Asp Ser 85 90 95 Asp Ser Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser 100 105 110 Asp Ser Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser 115 120 125 Asp Ser Asp Ser Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser 130 135 140 Asp Ser Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser 145 150 155 160 Asp Ser AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser 165 170 175 Asp SerAsp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser 180 185 190 AspSer Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser Asp Ser 195 200 205Asp Ser Gly Thr Ser Ser Gly Lys Gly Ser His Thr Gly Lys Lys Pro 210 215220 Gly Asn Pro Lys Gly Asn Thr Asn Arg Pro Ser Gln Arg His Thr Asn 225230 235 240 Gln Pro Gln Arg Pro Lys Tyr Asn Gln Thr Asn Gln Asn Asn IleAsn 245 250 255 Asn Ile Asn His Asn Ile Asn His Thr Arg Thr Ser Gly AspGly Ala 260 265 270 Pro Phe Lys Arg Gln Gln Asn Ile Ile Asn Ser Asn SerGly His Arg 275 280 285 Asn Gln Asn Asn Ile Asn Gln Phe Ile Trp Asn LysAsn Gly Phe Phe 290 295 300 Lys Ser Gln Asn Asn Thr Glu His Arg Met AsnSer Ser Asp Asn Thr 305 310 315 320 Asn Ser Leu Ile Ser Arg Phe Arg GlnLeu Ala Thr Gly Ala Tyr Lys 325 330 335 Tyr Asn Pro Phe Leu Ile Asn GlnVal Lys Asn Leu Asn Gln Leu Asp 340 345 350 Gly Lys Val Thr Asp Ser AspIle Tyr Ser Leu Phe Arg Lys Gln Ser 355 360 365 Phe Arg Gly Asn Glu TyrLeu Asn Ser Leu Gln Lys Gly Thr Ser Tyr 370 375 380 Phe Arg Phe Gln TyrPhe Asn Pro Leu Asn Ser Ser Lys Tyr Tyr Glu 385 390 395 400 Asn Leu AspAsp Gln Val Leu Ala Leu Ile Thr Gly Glu Ile Gly Ser 405 410 415 Met ProGlu Leu Lys Lys Pro Thr Asp Lys Glu Asp Lys Asn His Ser 420 425 430 AlaPhe Lys Asn His Ser Ala Asp Glu Ile Thr Thr Asn Asn Asp Gly 435 440 445His Ser Lys Asp Tyr Asp Lys Lys Lys Lys Ile His Arg Ser Leu Leu 450 455460 Ser Leu Ser Ile Ala Ile Ile Gly Ile Phe Leu Gly Val Thr Gly Leu 465470 475 480 Tyr Ile Phe Arg Arg Lys Lys 485 15 18 DNA Staphylococcusaureus misc_feature n=(a or c or g or t) 15 gaytcngayt cngayagy 18 16 5PRT Staphylococcus aureus 16 Leu Pro Asp Thr Gly 1 5 17 9 PRTStaphylococcus aureus 17 Thr Tyr Thr Phe Thr Asp Tyr Val Asp 1 5 18 6PRT Staphylococcus aureus 18 Thr Asn Ser His Gln Asp 1 5

What is claimed is:
 1. A multicomponent vaccine consisting essentiallyof immunologically effective amounts of the collagen binding domain of aStaphylococcal collagen binding protein, and the fibrinogen bindingdomain of a Staphylococcal fibrinogen binding protein, and apharmaceutically acceptable carrier or excipient.
 2. A vaccine accordingto claim 1 further comprising the fibronectin binding domain of aStaphylococcal fibronectin binding protein.
 3. A vaccine according toclaim 1 wherein the staphylococcal organisms are Staphylococcus aureus.4. A vaccine according to claim 3, wherein the Staphylococcus aureusbinding proteins are selected from the group consisting of the collagenbinding adhesin CNA, clumping factor A (ClfA) and clumping factor B(ClfB).
 5. A vaccine according to claim 1 further comprising an SdrHprotein from Staphylococcus epidermidis.
 6. A vaccine according to claim1 wherein the collagen binding protein is selected from the groupconsisting of the collagen binding adhesin CNA and the collagen bindingadhesin subdomain M55.
 7. A vaccine according to claim 1 wherein thefibrinogen binding protein is selected from the group consisting ofclumping factor A (ClfA) and clumping factor B (ClfB).
 8. A vaccineaccording to claim 2 wherein the fibronectin binding protein is selectedfrom the group consisting of fibronectin binding protein A (FnBP-A) andfibronectin binding protein B (FnBP-B).